3. Protein quantitation
• Protein quantitation is often necessary before and after
processing protein samples for isolation, separation and analysis
by chromatographic, electrophoretic and immunochemical
methods.
• Most colorimetric protein assay methods can be divided into two
groups.
– Protein-copper chelation: BCA (Bicinchoninic Acid) and modified
Lowry assay
– Protein-dye binding: Coomassie (Bradford) assay.
• Choice of assay: Buffer compatibility (the presence of reducing
agent and detergent in the protein solution may interfere
chemical reaction of protein quantitation)
3
4. Protein quantitation
• Protein standard curve: bovine serum albumin (BSA), known
concentration of protein standard to determine the
concentration of purified proteins by interpolation.
4
6. Peptide sequencing – N-terminal amino acid
analysis
• React the peptide with a reagent that will selectively label the
terminal amino acid.
• Hydrolyse the protein.
• Determine the amino acid by chromatography and comparison
with standards.
6
Sanger analysis: The protein is
hydrolyzed by 6M HCl first, the
N-terminal group of amino acids
are labeled with 1-fluoro-2,4-
dinitrobenzene (Sanger’s
reagent, DNFB). The labeled
amino acids are separated and
identified.
7. Peptide sequencing – N-terminal amino acid
analysis
• Edman degradation, developed by Pehr Edman, is a method of
sequencing amino acids in a peptide.
• In this method, the amino-terminal residue is labeled and
cleaved from the peptide without disrupting other peptide
bonds between other amino acid residues.
• Phenylisothiocyanate (PITC) reacts with the free N-terminus of
the chain to form PTC derivative.
• Then, under acidic (TFA, trifluroacetic acid) conditions, this PTC
derivative is cleaved as a thiazolinone derivative.
7
8. Edman degradation
• The thiazolinone derivative is then selectively extracted into an
organic solvent and treated with acid to form the more stable
phenylthiohydantoin (PTH)- amino acid derivative that can be
identified by using chromatography or electrophoresis.
• This procedure can then be repeated again to identify the next
amino acid.
• Development of automatic sequencer has speed up the process,
1 cycle can be carried out in less than 1 hour.
8
10. Edman degradation: Advantage and
Disadvantage
• A major drawback to this technique is that the peptides being
sequenced in this manner cannot have more than 50 residues.
• This is because the Edman degradation reaction is not 100%
efficient, meaning that the cleavage step does not occur every
time.
• However, this problem can be resolved by cleaving large peptides
into smaller peptides before proceeding with the reaction.
• It is able to accurately sequence up to 30 amino acids with 98%
efficiency per amino acid.
• Advantage: it only uses 10 - 100 picomoles of peptide for the
sequencing process
10
11. Protein sequencing strategies
• Polypeptides and proteins with greater than 50 amino acid
residues must be cut into smaller pieces for sequencing.
• Proteins can be cleaved at specific sites by chemical such as
cyanogen bromide (CNBr) or enzymatically with enzymes like
trypsin, or chymotrypsin.
11
15. Protein sequencing strategies
• Proteins with disulfide bonds:
Must be broken with reducing
agents such as mercaptoethanol
and modified to prevent re-
formation
• Large proteins (>500 a.a.) :
Often fragmented with two or
more different methods.
15
16. Immunology provides important techniques
with which to investigate proteins
An antibody (immunoglobulin, Ig) is a protein synthesized by
an animal (human) in response to the presence of a foreign
substance (antigen). It normally functions to protect the
animal from infection.
• Antibodies have high specificities & affinities to
antigens.
• They recognize a specific group or clusters of amino acids
on a large molecule called antigenic determinant or
epitope.
• From the large repertoire of antibody-producing cells, each
produces an antibody of a single specificity (polyclonal).
16
18. Types of antibodies
18
• Polyclonal antibody: Heterogeneous
mixtures of antibodies, each specific
for one of the various epitopes on
an antigen. Useful for detection of a
protein of low abundance, because
protein molecules can be bound by
more than one antibody at multiple
distinct antigenic sites
19. Types of antibodies
19
• Monoclonal antibody: Identical,
produced by clones of a single
antibody-producing cell, recognizing
one specific epitope. Highly
specific, useful when working with
impure proteins.
21. Application of monoclonal antibody
• Used in affinity chromatography to purify proteins
• Used in clinical laboratories
– Detection of blood isozymes in MI
– Screening donated blood for HIV, hepatitis etc
• Therapeutic agent
– Herceptin a drug used in breast cancer treatment
• Used as catalytic antibody due to their very specific
binding properties
• Detection of proteins by immunofluorescence,
Western blot, immunosorbent assay
21
22. Immunofluoroscence
• Immunofluorescence is a technique used for light
microscopy with a fluorescence microscope and is used primarily
on microbiological samples.
• This technique uses the specificity of antibodies to their antigen to
target fluorescent dyes to specific biomolecule targets within a
cell, and therefore allows visualisation of the distribution of the
target molecule through the sample.
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23. Immunosorbent assay - ELISA
ELISA: Enzyme-Linked Immunosorbent Assay
• Ab used as specific analytic reagents to quantify the
amount of a protein or other antigen.
• Enzyme reacts with a colorless substrate to produce a
colored product.
• Enzyme linked to specific Ab that recognizes a target
antigen.
• If antigen is present the Ab-enzyme complex will bind to it .
23
96-well microtiter plate
used in a typical ELISA assay
24. Immunosorbent assay - ELISA
ELISA: Enzyme-Linked Immunosorbent Assay
• If antigen is present the Ab-enzyme complex will bind to it
• Upon addition of substrate, the enzyme will catalyze the
reaction and genetrate the color product
• The presence of a color product indicate the presence of
antigen
• The amount of color product proportional to amount of
antigen
• Need low amount of protein/antigen
24
26. Indirect ELISA
To detect presence of antibody
Example:
• HIV test detects the presence of antibodies that recognizes
viral core protein, which is the antigen.
• In indirect ELISA, the antigen at the bottom of the well is
the viral core protein.
• Serum from patient is added to the well.
• If patient has Aids/infected with HIV, he/she will have Ab to
the viral core proteins.
• Ab from his/her sample will bind to the antigen at the
bottom of the well.
26
27. Sandwich ELISA
To detect presence of antigen
Example
• Can use to detect cardiac markers after heart attack
• Bind Ab to creatine kinase to the bottom of the well
• During heart attack, creatine kinase (CK) is released within
hours of heart attack by cardiac muscle cells
• The patients will have CK in the serum, CK will bind to the
AB at the bottom of the well
• A second Ab-labeled with enzyme that recognize CK will
bind to CK. Second AB has enzyme that can react with
substrate, and upon reaction elicit color production
27
28. Western blotting/Immunoblotting
28
• Analytical technique used to detect specific proteins in a sample
of tissue homogenate or extract.
• It uses gel electrophoresis to separate proteins and the proteins
are then transferred to a membrane (typically PVDF
or nitrocellulose), where they are detected
with antibodies specific to the target protein.
Detection: Indirect
method, secondary
antibody can
amplify the signal,
colorimetric or
chemiluminescent.
31. The 3D protein structure can be determined by
NMR spectroscopy, X-ray crystallography and
CryoEM
31
• Protein function depends on 3D structure.
Nuclear Magnetic
Resonance (NMR)
spectroscopy reveal the
dynamics of structure of
proteins in solution.
X-ray crystallography provide
the best resolution for the
determination of molecular
structures.
Cryo-electron microscopy
(cryo-EM), a technique
that fires beams of
electrons at proteins that
have been frozen in
solution, to deduce the
biomolecules’ structure.
32. Nuclear Magnetic Resonance
• NMR depends on the fact that
certain atomic nuclei are
intrinsically magnetic.
• The chemical shift of nuclei depends
on their local environment. The
spins of neighboring nuclei interact
with each other in ways that provide
definitive structural information.
32
33. X-ray crystallography
Preparation of a protein in crystal form
• Challenging process: A concentrated solution of highly pure
material is required. It is often difficult to predict which
experimental conditions will yield the most effective crystals.
• Protein frequently crystallize in their biologically active
configuration.
33
34. X-ray crystallography
• Provide the best resolution for
the determination of molecular
structures because their
wavelength approximately
corresponds to that of a covalent
bond.
• An x-ray source generates a
beam, which is diffracted by a
crystal. The resulting diffraction
pattern is collected on a detector.
34
35. X-ray crystallography
• A crystal is mounted and gradually
rotated while being bombarded
with X-rays, producing a diffraction
pattern.
• The two-dimensional images taken
at different rotations are converted
into a three-dimensional model,
combined with chemical data
known for the sample.
• The scattering pattern provide
abundant information about
protein structure.
35
36. Cryo-electron microscopy
• Electron microscopy (EM)
technique where the sample is
cooled to cryogenic temperatures.
• Limited information about
dynamics
• Can be used for very large
molecules and complexes
36
37. Visualizing Protein Structures
• The protein structure data are stored in Protein Data Bank
(http://www.pdb.org)
• The complexity of proteins with thousands of atoms presents a
challenge for the depiction of their structure.
• Several different types of representations are used to portray
proteins, each with its own strengths and weaknesses.
– Space-filling model
– Ball-and-stick model
– Ribbon diagrams
37
38. Space-filling model
• The most realistic type of representation
• Each atom is shown as a sphere with a
size corresponding to the van der Waals
radius of the atom. Bonds are not shown
explicitly.
• Advantage: useful in showing
conformational changes in a protein
from one set of circumstances to
another.
• Disadvantage: secondary and tertiary
structures are difficult to see.
38
39. Ball-and-stick models
• Not as realistic as space-filling models,
but the bonding arrangement is easier to
see because the bonds are explicitly
represented as sticks.
• Reveals a complex structure more clearly
than a space-filling model. However, the
depiction is so complicated that
structural features such a alpha helices
are difficult to discern.
• Because space-filling and ball-and-stick
models depict protein structures at the
atomic level, the large number of atoms
in a complex structure makes it difficult
to discern the relevant features.
39
40. Ribbon diagram
• Use schematic representation
instead, such as ribbon diagram.
• Used to accent a few dramatic
aspects of protein structures, such as
alpha helix (coiled ribbon), loop (thin
tube), beta strand (broad arrow).
• Allow the course of a polypeptide
chain to be traced and readily shows
the 2nd structure.
40
41. Ribbon diagram with highlights
• Protein structures are tightly packed and little open space.
• The openness of ribbon diagrams makes them particularly
useful to highlight additional aspects of protein structure.
• Active sites, substrates, bonds can be included in ball-and-
stick or space-filling form within a ribbon diagram.
41
42. Summary
1. The purification of a protein is an essential first step in
understanding their function.
2. Amino acid sequences of proteins can be determined
experimentally.
3. Immunology provides important techniques with which to
investigate proteins.
4. Mass spectrometry is a powerful technique for the identification
of peptides and proteins.
5. Three-dimensional protein structure can be determined by X-ray
crystallography, NMR spectroscopy and Cryo-EM.
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43. Study questions
1. How do you sequence protein with more than 50 amino acid
residues long?
2. What is the application of monoclonal antibody?
3. Name protein detection methods using immunological
approach.
4. What is a Western blotting?
5. What is the purpose of using indirect ELISA and sandwich ELISA?
6. Name the methods to determine protein 3D structure.
7. What are the commonly used models to visualize protein 3D
structure?
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