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Protein Analysis MethodsProtein Analysis Methods
andand ApplicationApplication
LECTURE BYLECTURE BY
ANGEL L. SALAMAN-BAYRO...
OUTLINEOUTLINE
 Gel ElectrophoresisGel Electrophoresis
 Gel Electrophoresis Under denaturingGel Electrophoresis Under de...
Gel ElectrophoresisGel Electrophoresis
 Gel electrophoresisGel electrophoresis is a technique used for theis a technique ...
Gel ElectrophoresisGel Electrophoresis
 Proteins, unlike nucleic acids, can have varying chargesProteins, unlike nucleic ...
SDS-PAGE (PolyAcrylamide GelSDS-PAGE (PolyAcrylamide Gel
Electrophoresis)Electrophoresis)
 The purpose of this method is ...
SDS-PAGE (PolyAcrylamide GelSDS-PAGE (PolyAcrylamide Gel
Electrophoresis)Electrophoresis)
 SDSSDS (sodium dodecyl sulfate...
SDS-PAGE (PolyAcrylamide GelSDS-PAGE (PolyAcrylamide Gel
Electrophoresis)Electrophoresis)
 The top portion of the figure ...
SDS-PAGE (PolyAcrylamide GelSDS-PAGE (PolyAcrylamide Gel
ElectrophoresisElectrophoresis
This Figure shows a slab of polyac...
Native" or "non-denaturing" gelNative" or "non-denaturing" gel
electrophoresiselectrophoresis
 ……is run in the absence of...
 Thus native gels can be sensitive to anyThus native gels can be sensitive to any
process that alters either the charge o...
Native" or "non-denaturing" gelNative" or "non-denaturing" gel
electrophoresiselectrophoresis
 These properties, and thei...
Native" or "non-denaturing" gelNative" or "non-denaturing" gel
electrophoresiselectrophoresis
It is therefore possible to ...
2D Gel Electrophoresis2D Gel Electrophoresis
 This is a method for the separation andThis is a method for the separation ...
2D Gel Electrophoresis2D Gel Electrophoresis
How is it Performed?How is it Performed?
 Isoelectric focusingIsoelectric fo...
2D Gel Electrophoresis2D Gel Electrophoresis
 Ispelectrofocusing (IEF)Ispelectrofocusing (IEF) pH gradients can be genera...
2D Gel Electrophoresis2D Gel Electrophoresis
 ……is generally used as a component of proteomics and is the stepis generall...
Proteins Colorimetric AssayProteins Colorimetric Assay
 Hartree-Lowry and Modified Lowry Protein Assays:Hartree-Lowry and...
Proteins Colorimetric AssayProteins Colorimetric Assay
 Biuret Protein Assay:Biuret Protein Assay: The principle of the B...
Proteins Colorimetric AssayProteins Colorimetric Assay
 Bradford protein assay:Bradford protein assay: The Bradford assay...
Proteins Colorimetric AssayProteins Colorimetric Assay
 Bicinchoninic Acid (BCA) Protein Assay (Smith):Bicinchoninic Acid...
Protein ImmunostainingProtein Immunostaining
 ……is a general term in biochemistry that appliesis a general term in bioche...
Protein ImmunostainingProtein Immunostaining
 ImmunohistochemistryImmunohistochemistry or IHC staining of tissue sections...
Protein ImmunostainingProtein Immunostaining
 Tissue preparation orTissue preparation or fixationfixation is essential fo...
Protein ImmunostainingProtein Immunostaining
 The detection of many antigens can be dramatically improved byThe detection...
Protein ImmunostainingProtein Immunostaining
 AA flow cytometerflow cytometer is a technology that allows a single cell t...
Flow cytometerFlow cytometer
Protein ImmunostainingProtein Immunostaining
 Western blottingWestern blotting allows the detection of specific proteinsa...
No bands present Neg
Bands at either p31 OR p24
AND bands present at
either gp160 OR gp120
Pos
Bands present, but pattern
...
Western BlottingWestern Blotting
 Western blotting transferWestern blotting transfer
apparatusapparatus.. Schematic showi...
Western BlottingWestern Blotting
Protein ImmunostainingProtein Immunostaining
 The enzyme-linked immunosorbent assay or ELISA is aThe enzyme-linked immuno...
Protein ImmunostainingProtein Immunostaining
                                           
Advantages
•Quicker since only on...
ELISA ASSAY PROCESS
Electron microscopyElectron microscopy
 ……or EM can be used to study the detailedor EM can be used to study the detailed
...
Protein Assays byProtein Assays by
SpectrophotometrySpectrophotometry
PrinciplesPrinciples
 A spectrophotometer consists ...
Protein Assays byProtein Assays by
SpectrophotometrySpectrophotometry
Principles (cont.)Principles (cont.)
 When monochro...
Protein Assays byProtein Assays by
SpectrophotometrySpectrophotometry
 Proteins in solution absorb ultraviolet lightProte...
Protein Assays byProtein Assays by
SpectrophotometrySpectrophotometry
 Unknown proteins or protein mixturesUnknown protei...
Enzyme AssaysEnzyme Assays
 ……are laboratory methods for measuring enzymatic activity. They are vital forare laboratory m...
Enzyme AssaysEnzyme Assays
Related terminologyRelated terminology
 TheThe rate of a reactionrate of a reaction is the con...
Types of Enzyme AssaysTypes of Enzyme Assays
 All enzyme assays measure either the consumption of substrate or production...
Types of Enzyme Assays (cont.)Types of Enzyme Assays (cont.)
 Transient kinetics experimentsTransient kinetics experiment...
Types of Enzyme Assays (cont.)Types of Enzyme Assays (cont.)
Direct versus coupled assaysDirect versus coupled assays
 Co...
Types of Continuous EnzymeTypes of Continuous Enzyme
AssaysAssays
 FluorometricFluorometric ((Fluorescence) is when a mol...
Types of Continuous EnzymeTypes of Continuous Enzyme
AssaysAssays
 CalorimetricCalorimetric ((Calorimetry) is the measure...
Types of Continuous EnzymeTypes of Continuous Enzyme
AssaysAssays
 Light ScatteringLight Scattering:: measures the produc...
Types of Discontinuous EnzymeTypes of Discontinuous Enzyme
AssaysAssays
 RadiometricRadiometric assays measure the incorp...
Factors to control in EnzymeFactors to control in Enzyme
AssaysAssays
 Salt Concentration:Salt Concentration: Most enzyme...
Factors to control in EnzymeFactors to control in Enzyme
AssaysAssays
 Effects of pH:Effects of pH: Most enzymes are sens...
Immune FluorescenceImmune Fluorescence
 Most commonly, immunofluorescence employs two sets ofMost commonly, immunofluores...
Immune FluorescenceImmune Fluorescence
 As with most fluorescence techniques, a significant problem withAs with most fluo...
Method overviewMethod overview
1.1. In immunostaining methods, anIn immunostaining methods, an antibodyantibody is used to...
Method overviewMethod overview
4.4. The primary antibody can be probed for using aThe primary antibody can be probed for u...
ApplicationsApplications
 The applications ofThe applications of immunostainingimmunostaining areare
numerous, but are mo...
Non-Traditional (Other) MethodsNon-Traditional (Other) Methods
 Mass Spectrometry:Mass Spectrometry: A mass spectrometer ...
Mass Spectrometer (MS)Mass Spectrometer (MS)
 All massAll mass
spectrometers consistspectrometers consist
of three distin...
Mass Spectrometer (MS)Mass Spectrometer (MS)
IonizerIonizer
 In the GC-MS discussed in this introduction, the charged par...
Mass Spectrometer (MS)Mass Spectrometer (MS)
Ion AnalyzerIon Analyzer
 Molecular ions and fragment ions are accelerated b...
Mass Spectrometer (MS)Mass Spectrometer (MS)
DetectorDetector
 There are many types of detectors, but most work byThere a...
Protein Structure PredictionProtein Structure Prediction
 is the prediction of the three-dimensionalis the prediction of ...
Protein SequencingProtein Sequencing
 Proteins are found in every cell and are essential to every biologicalProteins are ...
Protein SequencingProtein Sequencing
 Edman degradationEdman degradation: is a method of: is a method of
sequencing amino...
Protein SequencingProtein Sequencing
 PhenylisothiocyanatePhenylisothiocyanate is reacted with an uncharged terminal amin...
Protein SequencingProtein Sequencing
Protein Structural AlignmentsProtein Structural Alignments
 Structural alignmentStructural alignment is a form of sequenc...
Protein Structural AlignmentsProtein Structural Alignments
 Structural alignments can compare two sequences orStructural ...
Protein Structural AlignmentsProtein Structural Alignments
METHODS:METHODS:
 GANGSTAGANGSTA ((GGeneticenetic AAlgorithm f...
Protein Structural AlignmentsProtein Structural Alignments
METHODS:METHODS:
 MAMMOTH (MAMMOTH (MAtching Molecular Models ...
Protein Detection Methods and Application
Protein Detection Methods and Application
Protein Detection Methods and Application
Protein Detection Methods and Application
Protein Detection Methods and Application
Protein Detection Methods and Application
Protein Detection Methods and Application
Protein Detection Methods and Application
Protein Detection Methods and Application
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Protein Detection Methods and Application

Protein Detection Methods

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Protein Detection Methods and Application

  1. 1. Protein Analysis MethodsProtein Analysis Methods andand ApplicationApplication LECTURE BYLECTURE BY ANGEL L. SALAMAN-BAYRON, Ph.D.ANGEL L. SALAMAN-BAYRON, Ph.D.
  2. 2. OUTLINEOUTLINE  Gel ElectrophoresisGel Electrophoresis  Gel Electrophoresis Under denaturingGel Electrophoresis Under denaturing conditionsconditions  Gel Electrophoresis Under non-denaturingGel Electrophoresis Under non-denaturing conditionsconditions  2D Gel Electrophoresis2D Gel Electrophoresis  Colorimetric AssaysColorimetric Assays  Protein ImmunostainingProtein Immunostaining  Proteins Colorimetric AssayProteins Colorimetric Assay  ElectrofocusingElectrofocusing  Non-Traditional (Other) MethodsNon-Traditional (Other) Methods
  3. 3. Gel ElectrophoresisGel Electrophoresis  Gel electrophoresisGel electrophoresis is a technique used for theis a technique used for the separation of deoxyribonucleic acid (DNA),separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein moleculesribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix.using an electric current applied to a gel matrix. DNA Gel electrophoresis is generally only usedDNA Gel electrophoresis is generally only used after amplification of DNA via PCR. It is usuallyafter amplification of DNA via PCR. It is usually performed for analytical purposes, but may beperformed for analytical purposes, but may be used as a preparative technique prior to use ofused as a preparative technique prior to use of other methods such as mass spectrometry,other methods such as mass spectrometry, PCR, cloning, DNA sequencing, Southern andPCR, cloning, DNA sequencing, Southern and Western Blotting for further characterization.Western Blotting for further characterization.
  4. 4. Gel ElectrophoresisGel Electrophoresis  Proteins, unlike nucleic acids, can have varying chargesProteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate intoand complex shapes, therefore they may not migrate into the polyacryl amide gel at similar rates, or at all, whenthe polyacryl amide gel at similar rates, or at all, when placing a negative to positive EMF on the sample.placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presenceProteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulfate/of a detergent such as sodium dodecyl sulfate/ (SDS/SDP) that coats the proteins with a negative charge.(SDS/SDP) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the sizeGenerally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), soof the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overallthat the resulting denatured proteins have an overall negative charge, and all the proteins have a similarnegative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act likecharge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, thelong rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate inrate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge orthe gel is relative only to its size and not its charge or shape.shape.
  5. 5. SDS-PAGE (PolyAcrylamide GelSDS-PAGE (PolyAcrylamide Gel Electrophoresis)Electrophoresis)  The purpose of this method is to separateThe purpose of this method is to separate proteins according to their size, and noproteins according to their size, and no other physical feature. In order toother physical feature. In order to understand how this works, we have tounderstand how this works, we have to understand the two halves of the name:understand the two halves of the name: SDSSDS andand PAGEPAGE  SDS:SDS: Sodium Dodecyl SulfateSodium Dodecyl Sulfate  PAGE:PAGE: Polyacrylamide Gel ElectrophoresisPolyacrylamide Gel Electrophoresis
  6. 6. SDS-PAGE (PolyAcrylamide GelSDS-PAGE (PolyAcrylamide Gel Electrophoresis)Electrophoresis)  SDSSDS (sodium dodecyl sulfate) is a detergent(sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules(soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfbut also has a negative charge (sulfATEATE)) attached to it. Therefore, if a cell is incubatedattached to it. Therefore, if a cell is incubated with SDS, the membranes will be dissolved andwith SDS, the membranes will be dissolved and the proteins will be solubilized by the detergent,the proteins will be solubilized by the detergent, plus all the proteins will be covered with manyplus all the proteins will be covered with many negative charges. The end result has twonegative charges. The end result has two important features:important features:  1) all proteins contain only primary structure and1) all proteins contain only primary structure and  2) all proteins have a large negative charge which2) all proteins have a large negative charge which means they will all migrate towards the positive polemeans they will all migrate towards the positive pole when placed in an electric fieldwhen placed in an electric field
  7. 7. SDS-PAGE (PolyAcrylamide GelSDS-PAGE (PolyAcrylamide Gel Electrophoresis)Electrophoresis)  The top portion of the figure showsThe top portion of the figure shows a protein with negative and positivea protein with negative and positive charges due to the charged R-charges due to the charged R- groups of the particular amino acidsgroups of the particular amino acids in the protein. The large Hin the protein. The large H represents hydrophobic domainsrepresents hydrophobic domains where nonpolar R-groups havewhere nonpolar R-groups have collected in an attempt to get awaycollected in an attempt to get away from the polar water that surroundsfrom the polar water that surrounds the protein. The bottom portionthe protein. The bottom portion shows that SDS can break upshows that SDS can break up hydrophobic areas and coathydrophobic areas and coat proteins with many negativeproteins with many negative charges which overwhelms anycharges which overwhelms any positive charge in the protein due topositive charge in the protein due to positively charged R-groups. Thepositively charged R-groups. The resulting protein has beenresulting protein has been denatured by SDS (reduced to itsdenatured by SDS (reduced to its primary structure) and as a resultprimary structure) and as a result has been linearized.has been linearized.
  8. 8. SDS-PAGE (PolyAcrylamide GelSDS-PAGE (PolyAcrylamide Gel ElectrophoresisElectrophoresis This Figure shows a slab of polyacrylamide (dark gray) with tunnels (different sized red rings with shading to depict depth) exposed on the edge. Notice that there are many different sizes of tunnels scattered randomly throughout the gel.
  9. 9. Native" or "non-denaturing" gelNative" or "non-denaturing" gel electrophoresiselectrophoresis  ……is run in the absence of SDS. While in SDS-PAGE theis run in the absence of SDS. While in SDS-PAGE the electrophoretic mobility of proteins depends primarily on theirelectrophoretic mobility of proteins depends primarily on their molecular mass, in native PAGE the mobility depends on both themolecular mass, in native PAGE the mobility depends on both the protein's charge and its hydrodynamic size.protein's charge and its hydrodynamic size.  The electric charge driving the electrophoresis is governed by theThe electric charge driving the electrophoresis is governed by the intrinsic charge on the protein at the pH of the running buffer. Thisintrinsic charge on the protein at the pH of the running buffer. This charge will, of course, depend on the amino acid composition of thecharge will, of course, depend on the amino acid composition of the protein as well as post-translational modifications such as additionprotein as well as post-translational modifications such as addition of sialic acids.of sialic acids.  Since the protein retains its folded conformation, its hydrodynamicSince the protein retains its folded conformation, its hydrodynamic size and mobility on the gel will also vary with the nature of thissize and mobility on the gel will also vary with the nature of this conformation (higher mobility for more compact conformations,conformation (higher mobility for more compact conformations, lower for larger structures like oligomers). If native PAGE is carriedlower for larger structures like oligomers). If native PAGE is carried out near neutral pH to avoid acid or alkaline denaturation, then itout near neutral pH to avoid acid or alkaline denaturation, then it can be used to study conformation, self-association or aggregation,can be used to study conformation, self-association or aggregation, and the binding of other proteins or compounds.and the binding of other proteins or compounds.
  10. 10.  Thus native gels can be sensitive to anyThus native gels can be sensitive to any process that alters either the charge or theprocess that alters either the charge or the conformation of a protein. This makesconformation of a protein. This makes them excellent tools for detecting thingsthem excellent tools for detecting things such as:such as:  changes in charge due to chemical degradation (changes in charge due to chemical degradation (e.g.e.g. deamidation)deamidation)  unfolded, "molten globule", or other modifiedunfolded, "molten globule", or other modified conformationsconformations  oligomers and aggregates (both covalent and non-oligomers and aggregates (both covalent and non- covalent)covalent)  binding events (protein-protein or protein-ligand)binding events (protein-protein or protein-ligand) Native" or "non-denaturing" gelNative" or "non-denaturing" gel electrophoresiselectrophoresis
  11. 11. Native" or "non-denaturing" gelNative" or "non-denaturing" gel electrophoresiselectrophoresis  These properties, and their relatively high throughput,These properties, and their relatively high throughput, make native gels excellent tools for analyzingmake native gels excellent tools for analyzing accelerated stability samples, demonstratingaccelerated stability samples, demonstrating comparability of different lots or processes, or examiningcomparability of different lots or processes, or examining the effects of excipientsthe effects of excipients..  Another advantage of native gels is that it is possible toAnother advantage of native gels is that it is possible to recover proteins in their native state after the separation.recover proteins in their native state after the separation. Recovery of active biological materials may, however,Recovery of active biological materials may, however, need to be done prior to any fixing or staining.need to be done prior to any fixing or staining.  Today many labs unfortunately ignore this valuable toolToday many labs unfortunately ignore this valuable tool because they think native gels are just too hard to use,because they think native gels are just too hard to use, or because they mistakenly believe they can only beor because they mistakenly believe they can only be used with acidic proteins. Bused with acidic proteins. Bothoth basic and acidic proteins,basic and acidic proteins, starting with commercial pre-cast gels and changing thestarting with commercial pre-cast gels and changing the buffers can be run.buffers can be run.
  12. 12. Native" or "non-denaturing" gelNative" or "non-denaturing" gel electrophoresiselectrophoresis It is therefore possible to screen conditions that minimize such oligomer formation using native PAGE. In fact, the figure above shows no dimer formation in histidine, glycine or Tris-HCl buffers (all at 20 mM), consistent with the highly-reversible thermal unfolding of EPO in those conditions.
  13. 13. 2D Gel Electrophoresis2D Gel Electrophoresis  This is a method for the separation andThis is a method for the separation and identification of proteins in a sample byidentification of proteins in a sample by displacement in 2 dimensions oriented atdisplacement in 2 dimensions oriented at right angles to one another. This allowsright angles to one another. This allows the sample to separate over a larger area,the sample to separate over a larger area, increasing the resolution of eachincreasing the resolution of each component. A good, general, up to datecomponent. A good, general, up to date reference on this subject is Görg et al,reference on this subject is Görg et al, (2000).(2000).
  14. 14. 2D Gel Electrophoresis2D Gel Electrophoresis How is it Performed?How is it Performed?  Isoelectric focusingIsoelectric focusing (IEF) is used in the 1st(IEF) is used in the 1st Dimension (Righetti, P.G., 1983). This separatesDimension (Righetti, P.G., 1983). This separates proteins by their charge (pI).proteins by their charge (pI).  SDS-PAGESDS-PAGE in the 2nd Dimension. Thisin the 2nd Dimension. This separates proteins by their size (molecularseparates proteins by their size (molecular weight, MW).weight, MW).  The procedure is known asThe procedure is known as ISO-DALTISO-DALT: iso for: iso for isoelectric focusing and dalt for dalton weightisoelectric focusing and dalt for dalton weight
  15. 15. 2D Gel Electrophoresis2D Gel Electrophoresis  Ispelectrofocusing (IEF)Ispelectrofocusing (IEF) pH gradients can be generatedpH gradients can be generated by adding ampholytes to an acrylamide gel. These are aby adding ampholytes to an acrylamide gel. These are a mixture of amphoteric species with a range of pI values.mixture of amphoteric species with a range of pI values. They are used for carrier ampholyte IEF and in this caseThey are used for carrier ampholyte IEF and in this case the gel may be pre-focused before sample application.the gel may be pre-focused before sample application.  Ampholyte properties:Ampholyte properties:  Even conductivityEven conductivity  High buffering capacityHigh buffering capacity  Soluble at isoelectric pointSoluble at isoelectric point  Minimum interaction with focused proteinsMinimum interaction with focused proteins  ImmobilinesImmobilines, s, similar to Ampholytes but have beenimilar to Ampholytes but have been immobilised within the polyacrylamide gel producing animmobilised within the polyacrylamide gel producing an immobilised pH gradient or IPG that does not need to beimmobilised pH gradient or IPG that does not need to be pre-focused.pre-focused.
  16. 16. 2D Gel Electrophoresis2D Gel Electrophoresis  ……is generally used as a component of proteomics and is the stepis generally used as a component of proteomics and is the step used for the isolation of proteins for further characterization by massused for the isolation of proteins for further characterization by mass spectroscopy. Purposes, firstly for thespectroscopy. Purposes, firstly for the large scale identificationlarge scale identification of allof all proteins in a sample. This is undertaken when the global proteinproteins in a sample. This is undertaken when the global protein expression of an organism or a tissue is being investigated and isexpression of an organism or a tissue is being investigated and is best carried out on model organisms whose genomes have beenbest carried out on model organisms whose genomes have been fully sequenced. In this way the individual proteins can be morefully sequenced. In this way the individual proteins can be more readily identified from the mass spectrometry data. The second usereadily identified from the mass spectrometry data. The second use of this technique is differential expression, this is when you compareof this technique is differential expression, this is when you compare two or more samples to find differences in theirtwo or more samples to find differences in their protein expressionprotein expression.. For instance, you may be looking at drugs resistance in a parasite.For instance, you may be looking at drugs resistance in a parasite. In this case you might like to compare a resistant organism to aIn this case you might like to compare a resistant organism to a susceptible one in an attempt to find the changes responsible for thesusceptible one in an attempt to find the changes responsible for the resistance. Here the sequence requirements of the organism are notresistance. Here the sequence requirements of the organism are not as important, as you are looking for a relatively small number ofas important, as you are looking for a relatively small number of differences and so can devote more time to the identification of eachdifferences and so can devote more time to the identification of each protein.protein.
  17. 17. Proteins Colorimetric AssayProteins Colorimetric Assay  Hartree-Lowry and Modified Lowry Protein Assays:Hartree-Lowry and Modified Lowry Protein Assays: The Lowry assayThe Lowry assay (1951) is an often-cited general use protein assay. For some time it(1951) is an often-cited general use protein assay. For some time it was the method of choice for accurate protein determination for cellwas the method of choice for accurate protein determination for cell fractions, chromatography fractions, enzyme preparations, and sofractions, chromatography fractions, enzyme preparations, and so on. The bicinchoninic acid (BCA) assay is based on the sameon. The bicinchoninic acid (BCA) assay is based on the same principle and can be done in one step, therefore it has beenprinciple and can be done in one step, therefore it has been suggested (Stoscheck, 1990) that the 2-step Lowry method issuggested (Stoscheck, 1990) that the 2-step Lowry method is outdated. However, the modified Lowry is done entirely at roomoutdated. However, the modified Lowry is done entirely at room temperature. The Hartree version of the Lowry assay, a more recenttemperature. The Hartree version of the Lowry assay, a more recent modification that uses fewer reagents, improves the sensitivity withmodification that uses fewer reagents, improves the sensitivity with some proteins, is less likely to be incompatible with some saltsome proteins, is less likely to be incompatible with some salt solutions, provides a more linear response, and is less likely tosolutions, provides a more linear response, and is less likely to become saturated.become saturated.  Reaction Principle:Reaction Principle: Under alkaline conditions the divalent copper ionUnder alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to aforms a complex with peptide bonds in which it is reduced to a monovalent ion. Monovalent copper ion and the radical groups ofmonovalent ion. Monovalent copper ion and the radical groups of tyrosine, tryptophan, and cysteine react with Folin reagent totyrosine, tryptophan, and cysteine react with Folin reagent to produce an unstable product that becomes reduced toproduce an unstable product that becomes reduced to molybdenum/tungsten bluemolybdenum/tungsten blue
  18. 18. Proteins Colorimetric AssayProteins Colorimetric Assay  Biuret Protein Assay:Biuret Protein Assay: The principle of the Biuret assayThe principle of the Biuret assay is similar to that of the Lowry, however it involves ais similar to that of the Lowry, however it involves a single incubation of 20 min. There are very fewsingle incubation of 20 min. There are very few interfering agents (ammonium salts being one suchinterfering agents (ammonium salts being one such agent), and Layne (1957) reported fewer deviations thanagent), and Layne (1957) reported fewer deviations than with the Lowry or ultraviolet absorption methods.with the Lowry or ultraviolet absorption methods. However, the biuret assay consumes much moreHowever, the biuret assay consumes much more material. The biuret is a good general protein assay formaterial. The biuret is a good general protein assay for batches of material for which yield is not a problem. Thebatches of material for which yield is not a problem. The Bradford assay is faster and more sensitive.Bradford assay is faster and more sensitive.  Reaction Principle:Reaction Principle: Under alkaline conditionsUnder alkaline conditions substances containing two or more peptide bonds form asubstances containing two or more peptide bonds form a purple complex with copper salts in the reagentpurple complex with copper salts in the reagent
  19. 19. Proteins Colorimetric AssayProteins Colorimetric Assay  Bradford protein assay:Bradford protein assay: The Bradford assay is very fast and uses about theThe Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It is fairly accurate and samplessame amount of protein as the Lowry assay. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford isthat are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content ofrecommended for general use, especially for determining protein content of cell fractions and assessing protein concentrations for gel electrophoresis.cell fractions and assessing protein concentrations for gel electrophoresis.  Assay materials including color reagent, protein standard, and instructionAssay materials including color reagent, protein standard, and instruction booklet are available from Bio-Rad Corporation. The method described belowbooklet are available from Bio-Rad Corporation. The method described below is for a 100 µl sample volume using 5 ml color reagent. It is sensitive to aboutis for a 100 µl sample volume using 5 ml color reagent. It is sensitive to about 5 to 200 micrograms protein, depending on the dye quality. In assays using 55 to 200 micrograms protein, depending on the dye quality. In assays using 5 ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µgml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µg protein. Scale down the volume for the "microassay procedure," which uses 1protein. Scale down the volume for the "microassay procedure," which uses 1 ml cuvettes. Protocols, including use of microtiter plates are described in theml cuvettes. Protocols, including use of microtiter plates are described in the flyer that comes with the Bio-Rad kit.flyer that comes with the Bio-Rad kit.  Reaction Principle:Reaction Principle: The assay is based on the observation that theThe assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-absorbance maximum for an acidic solution of Coomassie Brilliant Blue G- 250 shifts from 465 nm to 595 nm when binding to protein occurs. Both250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye,hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinctioncausing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-foldcoefficient of a dye-albumin complex solution is constant over a 10-fold concentration range.concentration range.
  20. 20. Proteins Colorimetric AssayProteins Colorimetric Assay  Bicinchoninic Acid (BCA) Protein Assay (Smith):Bicinchoninic Acid (BCA) Protein Assay (Smith): TheThe bicinchoninic acid (BCA) assay is available in kit form from Piercebicinchoninic acid (BCA) assay is available in kit form from Pierce (Rockford, Ill.). This procedure is very applicable to microtiter plate(Rockford, Ill.). This procedure is very applicable to microtiter plate methods. The BCA is used for the same reasons the Lowry is used.methods. The BCA is used for the same reasons the Lowry is used. Stoscheck (1990) has suggested that the BCA assay will replaceStoscheck (1990) has suggested that the BCA assay will replace the Lowry because it requires a single step, and the color reagent isthe Lowry because it requires a single step, and the color reagent is stable under alkaline conditions.stable under alkaline conditions.  Both a standard assay for concentrated proteins and a micro assay forBoth a standard assay for concentrated proteins and a micro assay for dilute protein solutions are described below.dilute protein solutions are described below.  Reaction Principle:Reaction Principle: BCA serves the purpose of the Folin reagent inBCA serves the purpose of the Folin reagent in the Lowry assay, namely to react with complexes between copperthe Lowry assay, namely to react with complexes between copper ions and peptide bonds to produce a purple end product. Theions and peptide bonds to produce a purple end product. The advantage of BCA is that the reagent is fairly stable under alkalineadvantage of BCA is that the reagent is fairly stable under alkaline conditions, and can be included in the copper solution to allow a oneconditions, and can be included in the copper solution to allow a one step procedure. A molybdenum/tungsten blue product is producedstep procedure. A molybdenum/tungsten blue product is produced as with the Lowry.as with the Lowry.
  21. 21. Protein ImmunostainingProtein Immunostaining  ……is a general term in biochemistry that appliesis a general term in biochemistry that applies to any use of an antibody-based method toto any use of an antibody-based method to detect a specific protein in a sample. The termdetect a specific protein in a sample. The term immunostaining was originally used to refer toimmunostaining was originally used to refer to the immunohistochemical staining of tissuethe immunohistochemical staining of tissue sections, as first described by Albert Coons insections, as first described by Albert Coons in 1941.Now however, immunostaining1941.Now however, immunostaining encompasses a broad range of techniques usedencompasses a broad range of techniques used in histology, cell biology, and molecular biologyin histology, cell biology, and molecular biology that utilize antibody-based staining methods.that utilize antibody-based staining methods.
  22. 22. Protein ImmunostainingProtein Immunostaining  ImmunohistochemistryImmunohistochemistry or IHC staining of tissue sectionsor IHC staining of tissue sections (or immunocytochemistry, which is the staining of cells),(or immunocytochemistry, which is the staining of cells), is perhaps the most commonly applied immunostainingis perhaps the most commonly applied immunostaining technique. While the first cases of IHC staining usedtechnique. While the first cases of IHC staining used fluorescent dyes, other non-fluorescent methods usingfluorescent dyes, other non-fluorescent methods using enzymes such as peroxidase and alkaline phosphataseenzymes such as peroxidase and alkaline phosphatase are now used. These enzymes are capable of catalysingare now used. These enzymes are capable of catalysing reactions that give a colored product that is easilyreactions that give a colored product that is easily detectable by light microscopy. Alternatively, radioactivedetectable by light microscopy. Alternatively, radioactive elements can be used as labels, and theelements can be used as labels, and the immunoreaction can be visualized by autoradiography.immunoreaction can be visualized by autoradiography.
  23. 23. Protein ImmunostainingProtein Immunostaining  Tissue preparation orTissue preparation or fixationfixation is essential for the preservation of cellis essential for the preservation of cell morphology and tissue architecture.morphology and tissue architecture.  Inappropriate or prolonged fixation may significantly diminish the antibodyInappropriate or prolonged fixation may significantly diminish the antibody binding capability.binding capability.  Many antigens can be successfully demonstrated in formalin-fixedMany antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections. However, some antigens will notparaffin-embedded tissue sections. However, some antigens will not survive even moderate amounts of aldehyde fixation. Under thesesurvive even moderate amounts of aldehyde fixation. Under these conditions, tissues should be rapidly fresh frozen in liquid nitrogenconditions, tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat.and cut with a cryostat.  The disadvantages of frozen sections include poor morphology, poorThe disadvantages of frozen sections include poor morphology, poor resolution at higher magnifications, difficulty in cutting over paraffinresolution at higher magnifications, difficulty in cutting over paraffin sections, and the need for frozen storage.sections, and the need for frozen storage.  Alternatively, vibratome sections do not require the tissue to beAlternatively, vibratome sections do not require the tissue to be processed through organic solvents or high heat, which can destroyprocessed through organic solvents or high heat, which can destroy the antigenicity, or disrupted by freeze thawing.the antigenicity, or disrupted by freeze thawing.  The disadvantage of vibratome sections is that the sectioning process isThe disadvantage of vibratome sections is that the sectioning process is slow and difficult with soft and poorly fixed tissues, and that chatter marksslow and difficult with soft and poorly fixed tissues, and that chatter marks or vibratome lines are often apparent in the sections.or vibratome lines are often apparent in the sections.
  24. 24. Protein ImmunostainingProtein Immunostaining  The detection of many antigens can be dramatically improved byThe detection of many antigens can be dramatically improved by antigen retrievalantigen retrieval methods that act by breaking some of the proteinmethods that act by breaking some of the protein cross-links formed by fixation to uncover hidden antigenic sites. Thiscross-links formed by fixation to uncover hidden antigenic sites. This can be accomplished by heating for varying lengths of times (heatcan be accomplished by heating for varying lengths of times (heat induced epitope retrieval or HIER) or using enzyme digestioninduced epitope retrieval or HIER) or using enzyme digestion (proteolytic induced epitope retrieval or PIER).(proteolytic induced epitope retrieval or PIER).  One of the main difficulties with IHC staining is overcoming specificOne of the main difficulties with IHC staining is overcoming specific or non-specific background. Optimisation of fixation methods andor non-specific background. Optimisation of fixation methods and times, pre-treatment with blocking agents, incubating antibodies withtimes, pre-treatment with blocking agents, incubating antibodies with high salt, and optimising post-antibody wash buffers and wash timeshigh salt, and optimising post-antibody wash buffers and wash times are all important for obtaining high quality immunostaining. Inare all important for obtaining high quality immunostaining. In addition, the presence of positive and negative controls for stainingaddition, the presence of positive and negative controls for staining are essential for determining specificity.are essential for determining specificity.
  25. 25. Protein ImmunostainingProtein Immunostaining  AA flow cytometerflow cytometer is a technology that allows a single cell to beis a technology that allows a single cell to be measured for a variety of characteristics, determined by looking atmeasured for a variety of characteristics, determined by looking at how they flow in liquid. Instruments used for this can gatherhow they flow in liquid. Instruments used for this can gather information about cells by measuring visible and fluorescent lightinformation about cells by measuring visible and fluorescent light emissions, allowing cell sorting based on physical, biochemical andemissions, allowing cell sorting based on physical, biochemical and antigenic traits.antigenic traits.  It can be used for the direct analysis of cells expressing one or moreIt can be used for the direct analysis of cells expressing one or more specific proteins. Cells are immunostained in solution usingspecific proteins. Cells are immunostained in solution using methods similar to used for immunofluorescence, and then analyzedmethods similar to used for immunofluorescence, and then analyzed by flow cytometry.by flow cytometry.  Flow cytometry has several advantages over IHC including: theFlow cytometry has several advantages over IHC including: the ability to define distinct cell populations are defined by their size andability to define distinct cell populations are defined by their size and granularity; the capacity to gate out dead cells; improved sensitivity;granularity; the capacity to gate out dead cells; improved sensitivity; and multi-colour analysis to measure several antigensand multi-colour analysis to measure several antigens simultaneously. However, flow cytometry can be less effective atsimultaneously. However, flow cytometry can be less effective at detecting extremely rare cell populations, and there is a loss ofdetecting extremely rare cell populations, and there is a loss of architectural relationships in the absence of a tissue section.architectural relationships in the absence of a tissue section.
  26. 26. Flow cytometerFlow cytometer
  27. 27. Protein ImmunostainingProtein Immunostaining  Western blottingWestern blotting allows the detection of specific proteinsallows the detection of specific proteins from extracts made from cells or tissues, before or afterfrom extracts made from cells or tissues, before or after any purification steps. Proteins are generally separatedany purification steps. Proteins are generally separated by size using gel electrophoresis before being transferredby size using gel electrophoresis before being transferred to a synthetic membrane via dry, semi-dry, or wet blottingto a synthetic membrane via dry, semi-dry, or wet blotting methods. The membrane can then be probed usingmethods. The membrane can then be probed using antibodies using methods similar toantibodies using methods similar to immunohistochemistry, but without a need for fixation.immunohistochemistry, but without a need for fixation. Detection is typically performed using peroxidase linkedDetection is typically performed using peroxidase linked antibodies to catalyse a chemiluminescent reaction.antibodies to catalyse a chemiluminescent reaction.  Western blotting is a routine molecular biology methodWestern blotting is a routine molecular biology method that can be used to semi-quantitatively compare proteinthat can be used to semi-quantitatively compare protein levels between extracts. The size separation prior tolevels between extracts. The size separation prior to blotting allows the protein molecular weight to be gaugedblotting allows the protein molecular weight to be gauged as compared with known molecular weight markersas compared with known molecular weight markers
  28. 28. No bands present Neg Bands at either p31 OR p24 AND bands present at either gp160 OR gp120 Pos Bands present, but pattern does not meet criteria for positivity +/- Western BlottingWestern Blotting
  29. 29. Western BlottingWestern Blotting  Western blotting transferWestern blotting transfer apparatusapparatus.. Schematic showingSchematic showing the assembly of a typicalthe assembly of a typical Western blot apparatus with theWestern blot apparatus with the position of the position of theposition of the position of the gel, transfer membrane andgel, transfer membrane and direction of protein in relation todirection of protein in relation to the electrode position. Althoughthe electrode position. Although the image depicted here isthe image depicted here is representative of a vertical "wet"representative of a vertical "wet" transfer apparatus, thetransfer apparatus, the orientation is applicable fororientation is applicable for horizontally positioned semi-dryhorizontally positioned semi-dry transfer apparatus.transfer apparatus.
  30. 30. Western BlottingWestern Blotting
  31. 31. Protein ImmunostainingProtein Immunostaining  The enzyme-linked immunosorbent assay or ELISA is aThe enzyme-linked immunosorbent assay or ELISA is a diagnostic method for quantitatively or semi-diagnostic method for quantitatively or semi- quantitatively determining protein concentrations fromquantitatively determining protein concentrations from blood plasma, serum or cell/tissue extracts in a multi-wellblood plasma, serum or cell/tissue extracts in a multi-well plate format (usually 96-wells per plate). Broadly,plate format (usually 96-wells per plate). Broadly, proteins in solution are adsorbed to ELISA plates.proteins in solution are adsorbed to ELISA plates. Antibodies specific for the protein of interest are used toAntibodies specific for the protein of interest are used to probe the plate. Background is minimized by optimizingprobe the plate. Background is minimized by optimizing blocking and washing methods (as for IHC), andblocking and washing methods (as for IHC), and specificity is ensured via the presence of positive andspecificity is ensured via the presence of positive and negative controls. Detection methods are usuallynegative controls. Detection methods are usually colorimetric or chemiluminescence based.colorimetric or chemiluminescence based.
  32. 32. Protein ImmunostainingProtein Immunostaining                                             Advantages •Quicker since only one antibody is used •No concern for cross-reactivity of a secondary antibody •Double possible with different labels on primary antibodies   Disadvantages •Labeling may reduce immunoreactivity of primary antibody •Labeled primary antibodies are expensive •Low flexibility in choice of primary antibody label •Little signal amplification                                                        Advantages •Secondary antibody can amplify signal •A variety of labeled secondary antibodies are available •One secondary may be used with many primary antibodies •Labeling does not affect primary antibody immunoreactivity •Changing secondary allows change of detection method Disadvantages •Secondary antibodies may produce nonspecific staining •Additional steps required compared to the direct method
  33. 33. ELISA ASSAY PROCESS
  34. 34. Electron microscopyElectron microscopy  ……or EM can be used to study the detailedor EM can be used to study the detailed microarchitecture of tissues or cells. Immuno-microarchitecture of tissues or cells. Immuno- EM allows the detection of specific proteins inEM allows the detection of specific proteins in ultrathin tissue sections. Antibodies labelled withultrathin tissue sections. Antibodies labelled with heavy metal particles (e.g. gold) can be directlyheavy metal particles (e.g. gold) can be directly visualised using transmission electronvisualised using transmission electron microscopy. While powerful in detecting the sub-microscopy. While powerful in detecting the sub- cellular localization of a protein, immuno-EM cancellular localization of a protein, immuno-EM can be technically challenging, expensive, andbe technically challenging, expensive, and require rigorous optimization of tissue fixationrequire rigorous optimization of tissue fixation and processing methods.and processing methods.
  35. 35. Protein Assays byProtein Assays by SpectrophotometrySpectrophotometry PrinciplesPrinciples  A spectrophotometer consists of two instruments, namely aA spectrophotometer consists of two instruments, namely a spectrometerspectrometer for producing light of any selected color (wavelength),for producing light of any selected color (wavelength), and aand a photometerphotometer for measuring the intensity of light. Thefor measuring the intensity of light. The instruments are arranged so that liquid in a cuvette can be placedinstruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount ofbetween the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. Thelight passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, normally aphotometer delivers a voltage signal to a display device, normally a galvanometer. The signal changes as the amount of light absorbedgalvanometer. The signal changes as the amount of light absorbed by the liquid changes.by the liquid changes.  If development of color is linked to the concentration of a substanceIf development of color is linked to the concentration of a substance in solution then that concentration can be measured by determiningin solution then that concentration can be measured by determining the extent of absorption of light at the appropriate wavelength. Forthe extent of absorption of light at the appropriate wavelength. For example hemoglobin appears red because the hemoglobin absorbsexample hemoglobin appears red because the hemoglobin absorbs blue and green light rays much more effectively than red. Theblue and green light rays much more effectively than red. The degree of absorbance of blue or green light is proportional to thedegree of absorbance of blue or green light is proportional to the concentration of hemoglobin.concentration of hemoglobin.
  36. 36. Protein Assays byProtein Assays by SpectrophotometrySpectrophotometry Principles (cont.)Principles (cont.)  When monochromatic light (light of a specific wavelength) passes through a solutionWhen monochromatic light (light of a specific wavelength) passes through a solution there is usually a quantitative relationship (Beer's law) between the solutethere is usually a quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light, that is,concentration and the intensity of the transmitted light, that is, I = II = Ioo X 10X 10--kc1kc1  wherewhere II sub 0 is the intensity of transmitted light using the pure solvent,sub 0 is the intensity of transmitted light using the pure solvent, II is theis the intensity of the transmitted light when the colored compound is added, c isintensity of the transmitted light when the colored compound is added, c is concentration of the colored compound,concentration of the colored compound, ll is the distance the light passes through theis the distance the light passes through the solution, and k is a constant. If the light pathsolution, and k is a constant. If the light path ll is a constant, as is the case with ais a constant, as is the case with a spectrophotometer, Beer's law may be written,spectrophotometer, Beer's law may be written, I / II / Ioo = 10= 10--kckc = T= T  wherewhere kk is a new constant andis a new constant and TT is the transmittance of the solution. There is ais the transmittance of the solution. There is a logarithmic relationship between transmittance and the concentration of the coloredlogarithmic relationship between transmittance and the concentration of the colored compound. Thus,compound. Thus, -log T-log T == log 1/Tlog 1/T == kckc = Optical Density (OD)= Optical Density (OD)  The O.D. is directly proportional to the concentration of the colored compound. MostThe O.D. is directly proportional to the concentration of the colored compound. Most spectrophotometers have a scale that reads both in O.D. (absorbance) units, which isspectrophotometers have a scale that reads both in O.D. (absorbance) units, which is a logarithmic scale, and in % transmittance, which is an arithmetic scale. Asa logarithmic scale, and in % transmittance, which is an arithmetic scale. As suggested by the above relationships, the absorbance scale is the most useful forsuggested by the above relationships, the absorbance scale is the most useful for colorimetric assayscolorimetric assays
  37. 37. Protein Assays byProtein Assays by SpectrophotometrySpectrophotometry  Proteins in solution absorb ultraviolet lightProteins in solution absorb ultraviolet light with absorbance maxima @ 280 and 200with absorbance maxima @ 280 and 200 nm. Amino acids with aromatic rings arenm. Amino acids with aromatic rings are the primary reason for the absorbancethe primary reason for the absorbance peak at 280 nm. Peptide bonds arepeak at 280 nm. Peptide bonds are primarily responsible for the peak at 200primarily responsible for the peak at 200 nm. Secondary, tertiary, and quaternarynm. Secondary, tertiary, and quaternary structure all affect absorbance, thereforestructure all affect absorbance, therefore factors such as pH, ionic strength, etc. canfactors such as pH, ionic strength, etc. can alter the absorbance spectrum.alter the absorbance spectrum.
  38. 38. Protein Assays byProtein Assays by SpectrophotometrySpectrophotometry  Unknown proteins or protein mixturesUnknown proteins or protein mixtures.. Use the followingUse the following formula to roughly estimate protein concentration. Pathformula to roughly estimate protein concentration. Path length for most spectrometers is 1 cm.length for most spectrometers is 1 cm.  ConcentrationConcentration (mg/ml) = Absorbance at 280 nm divided by path(mg/ml) = Absorbance at 280 nm divided by path length (cm.)length (cm.)  Pure protein of known absorbance coefficientPure protein of known absorbance coefficient.. Use theUse the following formula for a path length of 1 cm. Concentrationfollowing formula for a path length of 1 cm. Concentration is in mg/ml, %, or molarity depending on which typeis in mg/ml, %, or molarity depending on which type coefficient is used.coefficient is used.  ConcentrationConcentration = Absorbance at 280 nm divided by absorbance= Absorbance at 280 nm divided by absorbance coefficientcoefficient  To convert units, use these relationships:To convert units, use these relationships:  Mg protein/ml = % protein divided by 10 = molarity divided byMg protein/ml = % protein divided by 10 = molarity divided by protein molecular weightprotein molecular weight  Unknowns with possible nucleic acid contaminationUnknowns with possible nucleic acid contamination.. UseUse the following formula to estimate protein concentration:the following formula to estimate protein concentration:  Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)
  39. 39. Enzyme AssaysEnzyme Assays  ……are laboratory methods for measuring enzymatic activity. They are vital forare laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.the study of enzyme kinetics and enzyme inhibition.  Amounts of enzymes can either be expressed as molar amounts, as with anyAmounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of activity, in enzyme units.other chemical, or measured in terms of activity, in enzyme units.  Enzyme activityEnzyme activity = moles of substrate converted per unit time = rate ×= moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of activereaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions,enzyme present and is thus dependent on conditions, which should bewhich should be specifiedspecified. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively large unit. A more practical and commonly-used value is 1excessively large unit. A more practical and commonly-used value is 1 enzyme unit (EU) = 1 μmol min-1 (μ = micro, x 10-6). 1 U corresponds toenzyme unit (EU) = 1 μmol min-1 (μ = micro, x 10-6). 1 U corresponds to 16.67 nanokatals.16.67 nanokatals.  Specific activitySpecific activity of an enzyme is another common unit. This is the activity ofof an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol min-1mg-1).an enzyme per milligram of total protein (expressed in μmol min-1mg-1). Specific activity gives a measurement of the purity of the enzyme. It is theSpecific activity gives a measurement of the purity of the enzyme. It is the amount of product formed by an enzyme in a given amount of time underamount of product formed by an enzyme in a given amount of time under given conditions per milligram of enzyme. Specific activity is equal to the rategiven conditions per milligram of enzyme. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass ofof reaction multiplied by the volume of reaction divided by the mass of enzyme. The SI unit is katal kg-1, but a more practical unit is μmol mg-1 min-enzyme. The SI unit is katal kg-1, but a more practical unit is μmol mg-1 min- 1. Specific activity is a measure of enzyme processivity, usually constant for1. Specific activity is a measure of enzyme processivity, usually constant for a pure enzyme.a pure enzyme.
  40. 40. Enzyme AssaysEnzyme Assays Related terminologyRelated terminology  TheThe rate of a reactionrate of a reaction is the concentration ofis the concentration of substrate disappearing (or product produced)substrate disappearing (or product produced) per unit time (per unit time (molmol LL − 1− 1ss − 1)− 1)  TheThe % purity% purity is 100% × (specific activity ofis 100% × (specific activity of enzyme sample / specific activity of pureenzyme sample / specific activity of pure enzyme). The impure sample has lower specificenzyme). The impure sample has lower specific activity because some of the mass is not actuallyactivity because some of the mass is not actually enzyme. If the specific activity of 100% pureenzyme. If the specific activity of 100% pure enzyme is known, then an impure sample willenzyme is known, then an impure sample will have a lower specific activity, allowing purity tohave a lower specific activity, allowing purity to be calculated.be calculated.
  41. 41. Types of Enzyme AssaysTypes of Enzyme Assays  All enzyme assays measure either the consumption of substrate or production ofAll enzyme assays measure either the consumption of substrate or production of product over time. A large number of different methods of measuring theproduct over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed inconcentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalyzed reactions usingseveral different ways. Biochemists usually study enzyme-catalyzed reactions using four types of experiments:four types of experiments:  Initial rate experimentsInitial rate experiments. When an enzyme is mixed with a large excess of the. When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Thensubstrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediatesthe reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relativelyremains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steadyslowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because thestate, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excessmeasurements are carried out for a very short period and because of the large excess of substrate, the approximation free substrate is approximately equal to the initialof substrate, the approximation free substrate is approximately equal to the initial substrate can be made. The initial rate experiment is the simplest to perform andsubstrate can be made. The initial rate experiment is the simplest to perform and analyze, being relatively free from complications such as back-reaction and enzymeanalyze, being relatively free from complications such as back-reaction and enzyme degradation. It is therefore by far the most commonly used type of experiment indegradation. It is therefore by far the most commonly used type of experiment in enzyme kinetics.enzyme kinetics.  Progress curve experimentsProgress curve experiments.. In these experiments, the kinetic parameters areIn these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. Thedetermined from expressions for the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after the initial fastconcentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium.transient and for a sufficiently long period to allow the reaction to approach equilibrium. We note in passing that, while they are less common now, progress curve experimentsWe note in passing that, while they are less common now, progress curve experiments were widely used in the early period of enzyme kinetics.were widely used in the early period of enzyme kinetics.
  42. 42. Types of Enzyme Assays (cont.)Types of Enzyme Assays (cont.)  Transient kinetics experimentsTransient kinetics experiments. In these experiments, reaction behaviour. In these experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches theis tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to performsteady-state kinetics period. These experiments are more difficult to perform than either of the above two classes because they require rapid mixing andthan either of the above two classes because they require rapid mixing and observation techniques.observation techniques.  Relaxation experimentsRelaxation experiments. In these experiments, an equilibrium mixture of. In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature,enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored. The analysispressure or pH jump, and the return to equilibrium is monitored. The analysis of these experiments requires consideration of the fully reversible reaction.of these experiments requires consideration of the fully reversible reaction. Moreover, relaxation experiments are relatively insensitive to mechanisticMoreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism identification, althoughdetails and are thus not typically used for mechanism identification, although they can be under appropriate conditions.they can be under appropriate conditions.  Enzyme assays can be split into two groups according to their samplingEnzyme assays can be split into two groups according to their sampling method:method:  Continues assaysContinues assays where the assay gives a continuous reading of activity, andwhere the assay gives a continuous reading of activity, and  Discontinuous assaysDiscontinuous assays when samples are taken from an enzyme reaction atwhen samples are taken from an enzyme reaction at intervals and the amount of product production or substrate consumption isintervals and the amount of product production or substrate consumption is measured in these samples. Continuous assays are most convenient, with onemeasured in these samples. Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary.assay giving the rate of reaction with no further work necessary.
  43. 43. Types of Enzyme Assays (cont.)Types of Enzyme Assays (cont.) Direct versus coupled assaysDirect versus coupled assays  Coupled assay for hexokinase using glucose-6-Coupled assay for hexokinase using glucose-6- phosphate dehydrogenase.phosphate dehydrogenase.  Even when the enzyme reaction does not result in aEven when the enzyme reaction does not result in a change in the absorbance of light, it can still be possiblechange in the absorbance of light, it can still be possible to use a spectrophotometric assay for the enzyme byto use a spectrophotometric assay for the enzyme by using ausing a coupled assaycoupled assay. Here, the product of one. Here, the product of one reaction is used as the substrate of another, easily-reaction is used as the substrate of another, easily- detectable reaction. For example, figure 1 shows thedetectable reaction. For example, figure 1 shows the coupled assay for the enzyme hexokinase, which can becoupled assay for the enzyme hexokinase, which can be assayed by coupling its production of glucose-6-assayed by coupling its production of glucose-6- phosphate to NADPH production, using glucose-6-phosphate to NADPH production, using glucose-6- phosphate dehydrogenase.phosphate dehydrogenase.
  44. 44. Types of Continuous EnzymeTypes of Continuous Enzyme AssaysAssays  FluorometricFluorometric ((Fluorescence) is when a molecule emitsFluorescence) is when a molecule emits light of one wavelength after absorbing light of a differentlight of one wavelength after absorbing light of a different wavelength. Fluorometric assays use a difference in thewavelength. Fluorometric assays use a difference in the fluorescence of substrate from product to measure thefluorescence of substrate from product to measure the enzyme reaction. These assays are in general muchenzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but canmore sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and thesuffer from interference caused by impurities and the instability of many fluorescent compounds when exposedinstability of many fluorescent compounds when exposed to light.to light.  An example of these assays is again the use of the nucleotideAn example of these assays is again the use of the nucleotide coenzymes NADH and NADPH. Here, the reduced forms arecoenzymes NADH and NADPH. Here, the reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidationfluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease inreactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase. Syntheticfluorescence and reduction reactions by an increase. Synthetic substrates that release a fluorescent dye in an enzyme-catalyzedsubstrates that release a fluorescent dye in an enzyme-catalyzed reaction are also available, such as 4-methylumbelliferyl-β-D-reaction are also available, such as 4-methylumbelliferyl-β-D- galactoside for assaying β-galactosidase.galactoside for assaying β-galactosidase.
  45. 45. Types of Continuous EnzymeTypes of Continuous Enzyme AssaysAssays  CalorimetricCalorimetric ((Calorimetry) is the measurement of the heat releasedCalorimetry) is the measurement of the heat released or absorbed by chemical reactions. These assays are very general,or absorbed by chemical reactions. These assays are very general, since many reactions involve some change in heat and with use of asince many reactions involve some change in heat and with use of a microcalorimeter, not much enzyme or substrate is required. Thesemicrocalorimeter, not much enzyme or substrate is required. These assays can be used to measure reactions that are impossible toassays can be used to measure reactions that are impossible to assay in any other way.assay in any other way.  ChemiluminescentChemiluminescent ((Chemiluminescence) is the emission of lightChemiluminescence) is the emission of light by a chemical reaction. Some enzyme reactions produce light andby a chemical reaction. Some enzyme reactions produce light and this can be measured to detect product formation. These types ofthis can be measured to detect product formation. These types of assay can be extremely sensitive, since the light produced can beassay can be extremely sensitive, since the light produced can be captured by photographic film over days or weeks, but can be hardcaptured by photographic film over days or weeks, but can be hard to quantify, because not all the light released by a reaction will beto quantify, because not all the light released by a reaction will be detected.detected.  The detection of horseradish peroxidase by enzymaticThe detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method of detecting antibodieschemiluminescence (ECL) is a common method of detecting antibodies in western blotting. Another example is the enzyme luciferase, this isin western blotting. Another example is the enzyme luciferase, this is found in fireflies and naturally produces light from its substrate luciferin.found in fireflies and naturally produces light from its substrate luciferin.
  46. 46. Types of Continuous EnzymeTypes of Continuous Enzyme AssaysAssays  Light ScatteringLight Scattering:: measures the product ofmeasures the product of weight-averaged molar mass and concentrationweight-averaged molar mass and concentration of macromolecules in solution. Given a fixedof macromolecules in solution. Given a fixed total concentration of one or more species overtotal concentration of one or more species over the measurement time, the scattering signal is athe measurement time, the scattering signal is a direct measure of the weight-averaged molardirect measure of the weight-averaged molar mass of the solution, which will vary asmass of the solution, which will vary as complexes form or dissociate. Hence thecomplexes form or dissociate. Hence the measurement quantifies the stoichiometry of themeasurement quantifies the stoichiometry of the complexes as well as kinetics. Light scatteringcomplexes as well as kinetics. Light scattering assays of protein kinetics is a very generalassays of protein kinetics is a very general technique that does not require an enzyme.technique that does not require an enzyme.
  47. 47. Types of Discontinuous EnzymeTypes of Discontinuous Enzyme AssaysAssays  RadiometricRadiometric assays measure the incorporation of radioactivity intoassays measure the incorporation of radioactivity into substrates or its release from substrates. The radioactive isotopes mostsubstrates or its release from substrates. The radioactive isotopes most frequently used in these assays are 14C, 32P, 35S and 125I. Sincefrequently used in these assays are 14C, 32P, 35S and 125I. Since radioactive isotopes can allow the specific labeling of a single atom of aradioactive isotopes can allow the specific labeling of a single atom of a substrate, these assays are both extremely sensitive and specific. They aresubstrate, these assays are both extremely sensitive and specific. They are frequently used in biochemistry and are often the only way of measuring afrequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of enzymesspecific reaction in crude extracts (the complex mixtures of enzymes produced when you lyse cells).produced when you lyse cells).  Radioactivity is usually measured in these procedures using a scintillationRadioactivity is usually measured in these procedures using a scintillation counter.counter.  ChromatographicChromatographic assays measure product formation by separating theassays measure product formation by separating the reaction mixture into its components by chromatography. This is usuallyreaction mixture into its components by chromatography. This is usually done by high-performance liquid chromatography (HPLC), but can also usedone by high-performance liquid chromatography (HPLC), but can also use the simpler technique of thin layer chromatography. Although this approachthe simpler technique of thin layer chromatography. Although this approach can need a lot of material, its sensitivity can be increased by labeling thecan need a lot of material, its sensitivity can be increased by labeling the substrates/products with a radioactive or fluorescent tag. Assay sensitivitysubstrates/products with a radioactive or fluorescent tag. Assay sensitivity has also been increased by switching protocols to improvedhas also been increased by switching protocols to improved chromatographic instruments (e.g. ultra-high pressure liquidchromatographic instruments (e.g. ultra-high pressure liquid chromatography) that operate at pump pressure a few-fold higher thanchromatography) that operate at pump pressure a few-fold higher than HPLC instrumentsHPLC instruments
  48. 48. Factors to control in EnzymeFactors to control in Enzyme AssaysAssays  Salt Concentration:Salt Concentration: Most enzymes cannot tolerate extremely high saltMost enzymes cannot tolerate extremely high salt concentrations. The ions interfere with the weak ionic bonds of proteins.concentrations. The ions interfere with the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations of 1-500 mM. As usualTypical enzymes are active in salt concentrations of 1-500 mM. As usual there are exceptions such as the halophilic (salt loving) algae and bacteria.there are exceptions such as the halophilic (salt loving) algae and bacteria.  Effects of Temperature:Effects of Temperature: All enzymes work within a range of temperatureAll enzymes work within a range of temperature specific to the organism. Increases in temperature generally lead tospecific to the organism. Increases in temperature generally lead to increases in reaction rates. There is a limit to the increase because higherincreases in reaction rates. There is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates. This is due to thetemperatures lead to a sharp decrease in reaction rates. This is due to the denaturating (alteration) of protein structure resulting from the breakdown ofdenaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensionalthe weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme. The "optimum" temperature for human enzymes isstructure of the enzyme. The "optimum" temperature for human enzymes is usually between 35 and 40 °C. The average temperature for humans is 37usually between 35 and 40 °C. The average temperature for humans is 37 °C. Human enzymes start to denature quickly at temperatures above 40 °C.°C. Human enzymes start to denature quickly at temperatures above 40 °C.  Enzymes from thermophilic archaea found in the hot springs are stable up to 100Enzymes from thermophilic archaea found in the hot springs are stable up to 100 °C. However, the idea of an "optimum" rate of an enzyme reaction is misleading,°C. However, the idea of an "optimum" rate of an enzyme reaction is misleading, as the rate observed at any temperature is the product of two rates, the reactionas the rate observed at any temperature is the product of two rates, the reaction rate and the denaturation rate. If you were to use an assay measuring activity forrate and the denaturation rate. If you were to use an assay measuring activity for one second, it would give high activity at high temperatures, however if you wereone second, it would give high activity at high temperatures, however if you were to use an assay measuring product formation over an hour, it would give you lowto use an assay measuring product formation over an hour, it would give you low activity at these temperatures.activity at these temperatures.
  49. 49. Factors to control in EnzymeFactors to control in Enzyme AssaysAssays  Effects of pH:Effects of pH: Most enzymes are sensitive to pH and have specificMost enzymes are sensitive to pH and have specific ranges of activity. All have an optimum pH. The pH can stopranges of activity. All have an optimum pH. The pH can stop enzyme activity by denaturating (altering) the three dimensionalenzyme activity by denaturating (altering) the three dimensional shape of the enzyme by breaking ionic, and hydrogen bonds. Mostshape of the enzyme by breaking ionic, and hydrogen bonds. Most enzymes function between a pH of 6 and 8; however pepsin in theenzymes function between a pH of 6 and 8; however pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8.stomach works best at a pH of 2 and trypsin at a pH of 8.  Substrate Saturation:Substrate Saturation: Increasing the substrate concentrationIncreasing the substrate concentration increases the rate of reaction (enzyme activity). However, enzymeincreases the rate of reaction (enzyme activity). However, enzyme saturation limits reaction rates. An enzyme is saturated when thesaturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time. Atactive sites of all the molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no matter howthe saturation point, the reaction will not speed up, no matter how much additional substrate is added. The graph of the reaction ratemuch additional substrate is added. The graph of the reaction rate will plateau.will plateau.  Level of crowdingLevel of crowding, large amounts of macromolecules in a solution, large amounts of macromolecules in a solution will alter the rates and equilibrium constants of enzyme reactions,will alter the rates and equilibrium constants of enzyme reactions, through an effect called macromolecular crowding.through an effect called macromolecular crowding.
  50. 50. Immune FluorescenceImmune Fluorescence  Most commonly, immunofluorescence employs two sets ofMost commonly, immunofluorescence employs two sets of antibodies: a primary antibody is used against the antigen of interest;antibodies: a primary antibody is used against the antigen of interest; a subsequent, secondary ("indirect"), dye-coupled antibody isa subsequent, secondary ("indirect"), dye-coupled antibody is introduced that recognizes the primary antibody. In this fashion theintroduced that recognizes the primary antibody. In this fashion the researcher may create several primary antibodies that recognizeresearcher may create several primary antibodies that recognize various antigens, but, because they all share a common constantvarious antigens, but, because they all share a common constant region, may be recognized by a single dye-coupled antibody.region, may be recognized by a single dye-coupled antibody. Typically this is done by using antibodies made in different species.Typically this is done by using antibodies made in different species. For example, a researcher might create antibodies in a goat thatFor example, a researcher might create antibodies in a goat that recognize several antigens, and then employ dye-coupled rabbitrecognize several antigens, and then employ dye-coupled rabbit antibodies that recognize the goat antibody constant region (denotedantibodies that recognize the goat antibody constant region (denoted rabbit anti-goat). This allows re-use of the difficult-to-make dye-rabbit anti-goat). This allows re-use of the difficult-to-make dye- coupled antibodies in multiple experiments.coupled antibodies in multiple experiments.  In some cases, it is advantageous to use primary antibodies directlyIn some cases, it is advantageous to use primary antibodies directly labelled with a fluorophore. This direct labelling decreases thelabelled with a fluorophore. This direct labelling decreases the number of steps in the staining procedure and, more importantly,number of steps in the staining procedure and, more importantly, often avoids cross-reactivity and high background problems.often avoids cross-reactivity and high background problems. Fluorescent labelling can be performed in less than one hour withFluorescent labelling can be performed in less than one hour with readily available labeling kits.readily available labeling kits.
  51. 51. Immune FluorescenceImmune Fluorescence  As with most fluorescence techniques, a significant problem withAs with most fluorescence techniques, a significant problem with immunofluorescence is photobleaching. Loss of activity caused byimmunofluorescence is photobleaching. Loss of activity caused by photobleaching can be controlled by reducing the intensity or time-photobleaching can be controlled by reducing the intensity or time- span of light exposure, by increasing the concentration ofspan of light exposure, by increasing the concentration of fluorophores, or by employing more robust fluorophores that arefluorophores, or by employing more robust fluorophores that are less prone to bleaching (e.g. Alexa Fluors or DyLight Fluors).less prone to bleaching (e.g. Alexa Fluors or DyLight Fluors).  Many uses of immunofluorescence have been outmoded by theMany uses of immunofluorescence have been outmoded by the development of recombinant proteins containing fluorescent proteindevelopment of recombinant proteins containing fluorescent protein domains, e.g. green fluorescent protein (GFP). Use of such "tagged"domains, e.g. green fluorescent protein (GFP). Use of such "tagged" proteins allows much better localization and less disruption ofproteins allows much better localization and less disruption of protein function.protein function.
  52. 52. Method overviewMethod overview 1.1. In immunostaining methods, anIn immunostaining methods, an antibodyantibody is used tois used to detect a specificdetect a specific proteinprotein epitopeepitope. These antibodies can. These antibodies can bebe monoclonalmonoclonal oror polyclonalpolyclonal. Detection of this first or. Detection of this first or primary antibodyprimary antibody can be accomplished in multiplecan be accomplished in multiple ways.ways. 2.2. The primary antibody can be directly labeled using anThe primary antibody can be directly labeled using an enzymeenzyme oror fluorophorefluorophore.. 3.3. The primary antibody can be labeled using a smallThe primary antibody can be labeled using a small molecule which interacts with a high affinity bindingmolecule which interacts with a high affinity binding partner that can be linked to an enzyme or fluorophore.partner that can be linked to an enzyme or fluorophore. TheThe biotinbiotin-strepavidin is one commonly used high-strepavidin is one commonly used high affinity interaction.affinity interaction.
  53. 53. Method overviewMethod overview 4.4. The primary antibody can be probed for using aThe primary antibody can be probed for using a broader species-specificbroader species-specific secondary antibodysecondary antibody that isthat is labeled using an enzyme, or fluorophore.labeled using an enzyme, or fluorophore. 5.5. In the case ofIn the case of electron microscopyelectron microscopy, antibodies are, antibodies are linked to a heavy metallinked to a heavy metal atomatom.. 6.6. As previously described, enzymes such asAs previously described, enzymes such as horseradishhorseradish peroxidaseperoxidase oror alkalinealkaline phosphatasephosphatase are commonlyare commonly used to catalyse reactions that give a coloured orused to catalyse reactions that give a coloured or chemiluminescentchemiluminescent product. Fluorescent molecules canproduct. Fluorescent molecules can be visualised usingbe visualised using fluoresence microscopyfluoresence microscopy oror confocalconfocal microscopymicroscopy..
  54. 54. ApplicationsApplications  The applications ofThe applications of immunostainingimmunostaining areare numerous, but are most typically used innumerous, but are most typically used in clinicalclinical diagnosticsdiagnostics andand laboratory researchlaboratory research..  Clinically, IHC is used inClinically, IHC is used in histopathologyhistopathology for thefor the diagnosis of specific types of cancers based ondiagnosis of specific types of cancers based on molecular markers.molecular markers.  In laboratory science, immunostaining can beIn laboratory science, immunostaining can be used for a variety of applications based onused for a variety of applications based on investigating the presence or absence of ainvestigating the presence or absence of a protein, its tissue distribution, its sub-cellularprotein, its tissue distribution, its sub-cellular localization, and or changes in proteinlocalization, and or changes in protein expression or degradationexpression or degradation
  55. 55. Non-Traditional (Other) MethodsNon-Traditional (Other) Methods  Mass Spectrometry:Mass Spectrometry: A mass spectrometer creates charged particlesA mass spectrometer creates charged particles (ions) from molecules. It then analyzes those ions to provide(ions) from molecules. It then analyzes those ions to provide information about the molecular weight of the compound and itsinformation about the molecular weight of the compound and its chemical structure. There are many types of mass spectrometerschemical structure. There are many types of mass spectrometers and sample introduction techniques which allow a wide range ofand sample introduction techniques which allow a wide range of analyses. This discussion will focus on mass spectrometry as it'sanalyses. This discussion will focus on mass spectrometry as it's used in the powerful and widely used method of coupling Gasused in the powerful and widely used method of coupling Gas Chromatography (GC) with Mass Spectrometry (MS).Chromatography (GC) with Mass Spectrometry (MS).  Gas Chromatograph (GC):Gas Chromatograph (GC): A mixture of compounds to be analysedA mixture of compounds to be analysed is initially injected into the GC where the mixture is vaporized in ais initially injected into the GC where the mixture is vaporized in a heated chamber. The gas mixture travels through a GC column,heated chamber. The gas mixture travels through a GC column, where the compounds become separated as they interact with thewhere the compounds become separated as they interact with the column. The chromatogram on the right shows peaks which resultcolumn. The chromatogram on the right shows peaks which result from this separation. Those separated compounds then immediatelyfrom this separation. Those separated compounds then immediately enter the mass spectrometer.enter the mass spectrometer.
  56. 56. Mass Spectrometer (MS)Mass Spectrometer (MS)  All massAll mass spectrometers consistspectrometers consist of three distinctof three distinct regions.regions. 1) Ionizer1) Ionizer 2) Ion analyzer2) Ion analyzer 3) Detector3) Detector
  57. 57. Mass Spectrometer (MS)Mass Spectrometer (MS) IonizerIonizer  In the GC-MS discussed in this introduction, the charged particles (ions)In the GC-MS discussed in this introduction, the charged particles (ions) required for mass analysis are formed by Electron Impact (EI) Ionization.required for mass analysis are formed by Electron Impact (EI) Ionization. The gas molecules exiting the GC are bombarded by a high-energy electronThe gas molecules exiting the GC are bombarded by a high-energy electron beam (70 eV). An electron which strikes a molecule may impart enoughbeam (70 eV). An electron which strikes a molecule may impart enough energy to remove another electron from that molecule. Methanol, forenergy to remove another electron from that molecule. Methanol, for example, would undergo the following reaction in the ionizing region:example, would undergo the following reaction in the ionizing region: CH3OH + 1 electron CH3OH+CH3OH + 1 electron CH3OH+..+ 2 electrons+ 2 electrons (note: the symbols(note: the symbols +.+. indicate that a radical cation was formed)indicate that a radical cation was formed)  EI Ionization usually produces singly charged ions containing one unpairedEI Ionization usually produces singly charged ions containing one unpaired electron. A charged molecule which remains intact is called the molecularelectron. A charged molecule which remains intact is called the molecular ion. Energy imparted by the electron impact and, more importantly,ion. Energy imparted by the electron impact and, more importantly, instability in a molecular ion can cause that ion to break into smaller piecesinstability in a molecular ion can cause that ion to break into smaller pieces (fragments). The methanol ion may fragment in various ways, with one(fragments). The methanol ion may fragment in various ways, with one fragment carrying the charge and one fragment remaining uncharged. Forfragment carrying the charge and one fragment remaining uncharged. For example:example: CH3OH+CH3OH+..(molecular ion) CH2OH+(fragment ion) + H(molecular ion) CH2OH+(fragment ion) + H.. (or) CH3OH+(or) CH3OH+..(molecular ion) CH3+(fragment ion) +(molecular ion) CH3+(fragment ion) + ..OHOH
  58. 58. Mass Spectrometer (MS)Mass Spectrometer (MS) Ion AnalyzerIon Analyzer  Molecular ions and fragment ions are accelerated by manipulationMolecular ions and fragment ions are accelerated by manipulation of the charged particles through the mass spectrometer.of the charged particles through the mass spectrometer. Uncharged molecules and fragments are pumped away. TheUncharged molecules and fragments are pumped away. The quadrupole mass analyzer in this example uses positive (+) andquadrupole mass analyzer in this example uses positive (+) and negative (-) voltages to control the path of the ions. Ions travelnegative (-) voltages to control the path of the ions. Ions travel down the path based on their mass to charge ratio (m/z). EIdown the path based on their mass to charge ratio (m/z). EI ionization produces singly charged particles, so the charge (z) isionization produces singly charged particles, so the charge (z) is one. Therefore an ion's path will depend on its mass. If the (+) andone. Therefore an ion's path will depend on its mass. If the (+) and (-) rods shown in the mass spectrometer schematic were ?fixed' at a(-) rods shown in the mass spectrometer schematic were ?fixed' at a particular rf/dc voltage ratio, then one particular m/z would travel theparticular rf/dc voltage ratio, then one particular m/z would travel the successful path shown by the solid line to the detector. However,successful path shown by the solid line to the detector. However, voltages are not fixed, but are scanned so that ever increasingvoltages are not fixed, but are scanned so that ever increasing masses can find a successful path through the rods to the detector.masses can find a successful path through the rods to the detector.
  59. 59. Mass Spectrometer (MS)Mass Spectrometer (MS) DetectorDetector  There are many types of detectors, but most work byThere are many types of detectors, but most work by producing an electronic signal when struck by an ion.producing an electronic signal when struck by an ion. Timing mechanisms which integrate those signals withTiming mechanisms which integrate those signals with the scanning voltages allow the instrument to reportthe scanning voltages allow the instrument to report which m/z strikes the detector. The mass analyzer sortswhich m/z strikes the detector. The mass analyzer sorts the ions according to m/z and the detector records thethe ions according to m/z and the detector records the abundance of each m/z. Regular calibration of the m/zabundance of each m/z. Regular calibration of the m/z scale is necessary to maintain accuracy in thescale is necessary to maintain accuracy in the instrument. Calibration is performed by introducing ainstrument. Calibration is performed by introducing a well known compound into the instrument and "tweaking"well known compound into the instrument and "tweaking" the circuits so that the compound's molecular ion andthe circuits so that the compound's molecular ion and fragment ions are reported accurately.fragment ions are reported accurately.
  60. 60. Protein Structure PredictionProtein Structure Prediction  is the prediction of the three-dimensionalis the prediction of the three-dimensional structure of a protein from its amino acidstructure of a protein from its amino acid sequence—that is, the prediction of a protein'ssequence—that is, the prediction of a protein's tertiary structure from its primary structure. It istertiary structure from its primary structure. It is one of the most important goals pursued byone of the most important goals pursued by bioinformatics and theoretical chemistry. Proteinbioinformatics and theoretical chemistry. Protein structure prediction is of high importance instructure prediction is of high importance in medicine (for example, in drug design) andmedicine (for example, in drug design) and biotechnology (for example, in the design ofbiotechnology (for example, in the design of novel enzymes).novel enzymes).
  61. 61. Protein SequencingProtein Sequencing  Proteins are found in every cell and are essential to every biologicalProteins are found in every cell and are essential to every biological process, protein structure is very complex: determining a protein'sprocess, protein structure is very complex: determining a protein's structure involves firststructure involves first protein sequencingprotein sequencing - determining the amino- determining the amino acid sequences of its constituent peptides; and also determiningacid sequences of its constituent peptides; and also determining what conformation it adopts and whether it is complexed with anywhat conformation it adopts and whether it is complexed with any non-peptide molecules. Discovering the structures and functions ofnon-peptide molecules. Discovering the structures and functions of proteins in living organisms is an important tool for understandingproteins in living organisms is an important tool for understanding cellular processes, and allows drugs that target specific metaboliccellular processes, and allows drugs that target specific metabolic pathways to be invented more easily.pathways to be invented more easily.  The two major direct methods of protein sequencing areThe two major direct methods of protein sequencing are massmass spectrometryspectrometry and theand the Edman degradation reactionEdman degradation reaction. It is also. It is also possible to generate an amino acid sequence from the DNA orpossible to generate an amino acid sequence from the DNA or mRNA sequence encoding the protein, if this is known. However,mRNA sequence encoding the protein, if this is known. However, there are a number of other reactions which can be used to gainthere are a number of other reactions which can be used to gain more limited information about protein sequences and can be usedmore limited information about protein sequences and can be used as preliminaries to the aforementioned methods of sequencing or toas preliminaries to the aforementioned methods of sequencing or to overcome specific inadequacies within them.overcome specific inadequacies within them.
  62. 62. Protein SequencingProtein Sequencing  Edman degradationEdman degradation: is a method of: is a method of sequencing amino acids in a peptide. Insequencing amino acids in a peptide. In this method, the amino-terminal residue isthis method, the amino-terminal residue is labeled and cleaved from the peptidelabeled and cleaved from the peptide without disrupting the peptide bondswithout disrupting the peptide bonds between other amino acid residues.between other amino acid residues.
  63. 63. Protein SequencingProtein Sequencing  PhenylisothiocyanatePhenylisothiocyanate is reacted with an uncharged terminal aminois reacted with an uncharged terminal amino group, under mildly alkaline conditions, to form a cyclicalgroup, under mildly alkaline conditions, to form a cyclical phenylthiocarbamoylphenylthiocarbamoyl derivative. Then, under acidic conditions, thisderivative. Then, under acidic conditions, this derivative of the terminal amino acid is cleaved as a thiazolinonederivative of the terminal amino acid is cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extractedderivative. The thiazolinone amino acid is then selectively extracted into an organic solvent and treated with acid to form the more stableinto an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can bephenylthiohydantoin (PTH)- amino acid derivative that can be identified by using chromatography or electrophoresis.identified by using chromatography or electrophoresis.  This procedure can then be repeated again to identify the next aminoThis procedure can then be repeated again to identify the next amino acid. A major drawback to this technique is that the peptides beingacid. A major drawback to this technique is that the peptides being sequenced in this manner cannot have more than 50 to 60 residuessequenced in this manner cannot have more than 50 to 60 residues (and in practice, under 30). The peptide length is limited due to the(and in practice, under 30). The peptide length is limited due to the cyclical derivitization not always going to completion. Thecyclical derivitization not always going to completion. The derivitization problem can be resolved by cleaving large peptides intoderivitization problem can be resolved by cleaving large peptides into smaller peptides before proceeding with the reaction. It is able tosmaller peptides before proceeding with the reaction. It is able to accurately sequence up to 30 amino acids with modern machinesaccurately sequence up to 30 amino acids with modern machines capable of over 99% efficiency per amino acid. An advantage of thecapable of over 99% efficiency per amino acid. An advantage of the Edman degradation is that it only uses 10 - 100 picomoles of peptideEdman degradation is that it only uses 10 - 100 picomoles of peptide for the sequencing process. Edman degradation reaction isfor the sequencing process. Edman degradation reaction is automated to speed up the process.automated to speed up the process.
  64. 64. Protein SequencingProtein Sequencing
  65. 65. Protein Structural AlignmentsProtein Structural Alignments  Structural alignmentStructural alignment is a form of sequence alignment that is basedis a form of sequence alignment that is based on comparison of shape. These alignments attempt to establishon comparison of shape. These alignments attempt to establish equivalences between two or more polymer structures based onequivalences between two or more polymer structures based on their shape and three-dimensional conformation. This process istheir shape and three-dimensional conformation. This process is usually applied to protein tertiary structures but can also be used forusually applied to protein tertiary structures but can also be used for large RNA molecules. In contrast to simple structural superposition,large RNA molecules. In contrast to simple structural superposition, where at least some equivalent residues of the two structures arewhere at least some equivalent residues of the two structures are known, structural alignment requires noknown, structural alignment requires no a prioria priori knowledge ofknowledge of equivalent positions. Structural alignment is a valuable tool for theequivalent positions. Structural alignment is a valuable tool for the comparison of proteins with low sequence similarity, wherecomparison of proteins with low sequence similarity, where evolutionary relationships between proteins cannot be easilyevolutionary relationships between proteins cannot be easily detected by standard sequence alignment techniques. Structuraldetected by standard sequence alignment techniques. Structural alignment can therefore be used to imply evolutionary relationshipsalignment can therefore be used to imply evolutionary relationships between proteins that share very little common sequence. However,between proteins that share very little common sequence. However, caution should be used in using the results as evidence for sharedcaution should be used in using the results as evidence for shared evolutionary ancestry because of the possible confounding effectsevolutionary ancestry because of the possible confounding effects of convergent evolution by which multiple unrelated amino acidof convergent evolution by which multiple unrelated amino acid sequences converge on a common tertiary structure.sequences converge on a common tertiary structure.
  66. 66. Protein Structural AlignmentsProtein Structural Alignments  Structural alignments can compare two sequences orStructural alignments can compare two sequences or multiple sequencesmultiple sequences.. Because these alignments rely on information about all the queryBecause these alignments rely on information about all the query sequences' three-dimensional conformations, the method can only be usedsequences' three-dimensional conformations, the method can only be used on sequences where these structures are known. These are usually foundon sequences where these structures are known. These are usually found byby X-ray crystallographyX-ray crystallography oror NMR spectroscopyNMR spectroscopy. It is possible to perform a. It is possible to perform a structural alignment on structures produced bystructural alignment on structures produced by structure predictionstructure prediction methods.methods. Indeed, evaluating such predictions often requires a structural alignmentIndeed, evaluating such predictions often requires a structural alignment between the model and the true known structure to assess the model'sbetween the model and the true known structure to assess the model's quality. Structural alignments are especially useful in analyzing data fromquality. Structural alignments are especially useful in analyzing data from structuralstructural genomicsgenomics andand proteomicsproteomics efforts, and they can be used asefforts, and they can be used as comparison points to evaluate alignments produced by purely sequence-comparison points to evaluate alignments produced by purely sequence- basedbased bioinformaticsbioinformatics methods.methods.  The outputs of a structural alignment are a superposition of the atomicThe outputs of a structural alignment are a superposition of the atomic coordinate setscoordinate sets and a minimaland a minimal root mean squareroot mean square distance (distance (RMSDRMSD) between) between the structures. The RMSD of two aligned structures indicates theirthe structures. The RMSD of two aligned structures indicates their divergence from one another. Structural alignment can be complicated bydivergence from one another. Structural alignment can be complicated by the existence of multiplethe existence of multiple protein domainsprotein domains within one or more of the inputwithin one or more of the input structures, because changes in relative orientation of the domains betweenstructures, because changes in relative orientation of the domains between two structures to be aligned can artificially inflate the RMSD.two structures to be aligned can artificially inflate the RMSD.
  67. 67. Protein Structural AlignmentsProtein Structural Alignments METHODS:METHODS:  GANGSTAGANGSTA ((GGeneticenetic AAlgorithm forlgorithm for NNon-sequential,on-sequential, GGapped proteinapped protein STSTructureructure AAlignment) is a method forlignment) is a method for non-sequential protein structure alignment using a two-non-sequential protein structure alignment using a two- level hierarchical approach. On the first level, pairwiselevel hierarchical approach. On the first level, pairwise contacts and relative orientations between SSEs arecontacts and relative orientations between SSEs are maximized using a genetic algorithm (GA) and proteinmaximized using a genetic algorithm (GA) and protein graph representation. On the second level, pairwisegraph representation. On the second level, pairwise residue contact maps resulting from the best SSEresidue contact maps resulting from the best SSE alignments are optimized. GANGSTA can be used onlinealignments are optimized. GANGSTA can be used online atat http://agknapp.chemie.fu-berlin.de/gangstahttp://agknapp.chemie.fu-berlin.de/gangsta
  68. 68. Protein Structural AlignmentsProtein Structural Alignments METHODS:METHODS:  MAMMOTH (MAMMOTH (MAtching Molecular Models ObtainedMAtching Molecular Models Obtained from Theory)from Theory) -based structure alignment methods-based structure alignment methods decomposes the protein structure into short peptidesdecomposes the protein structure into short peptides (heptapeptides) which are compared with the(heptapeptides) which are compared with the heptapeptides of another protein. Similarity scoreheptapeptides of another protein. Similarity score between two heptapeptides is calculated using a unit-between two heptapeptides is calculated using a unit- vector RMS (URMS) method. These scores are stored invector RMS (URMS) method. These scores are stored in a similarity matrix, and with a hybrid (local-global)a similarity matrix, and with a hybrid (local-global) dynamic programming the optimal residue alignment isdynamic programming the optimal residue alignment is calculated. Protein similarity scores calculated withcalculated. Protein similarity scores calculated with MAMMOTH is derived from the likelihood of obtaining aMAMMOTH is derived from the likelihood of obtaining a given structural alignment by chancegiven structural alignment by chance

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