techniques of protein separation-aimec

1. Strategies
for
Protein Purification

2. Monomer Polymer Function(s)
Nucleotides DNA & RNA Genetic Information
Amino Acids Proteins Chemical
Reactions(Enzymes),
Structure
Sugars Polysaccharides Energy & Structure
Fatty Acids Lipids Membrane Structure &
Stability

3. Food rich in Proteins
We break down proteins in the food, using the building
blocks from those proteins to make new protein that cells
need, like antibody, hormones, etc.

4.  Protein purification is a series of processes intended to isolate a
single type of protein from a complex mixture.
 Protein purification is vital for the characterization of the
function, structure and interactions of the protein of interest.
 The starting material is usually a biological tissue or a
microbial culture.
 The various steps in the purification process may
free the protein from a matrix that confines
it, separate the protein and non-protein parts of the mixture,
and finally separate the desired protein from all other proteins.
The methods used in protein purification can roughly be divided
into analytical and preparative methods.

5. Protein Properties (Physical)
 Shape
 Sizes
 Density
 Charge

6. Separation of Amino Acids and Proteins
1.Ultracentrifugation –
Separation on the basis of molecular weight when large gravitational forces are
applied in the ultracentrifuge machine.
2-Ultrafiltration-Separation of solid from liquid under pressurefor smaller partical.
3.Chromatography –
The method of separating amino acids on the basis of differences
in absorption, ionic charges, size and solubility of molecules
4.Electrophoresis –
Separation in an electric field on the basis of differences in charges carried by
amino acids and proteins under specific condition
5.Precipitation Methods –
Salts as sodium sulfate, ammonium sulfate, cadmium nitrate, at specific conc.
precipitate some proteins while others remain in solution
6.Dialysis –It is for the removal of small, crystalloidal molecules from protein
solution through semipermeable membrane.

7.  An analytical purification generally utilizes three
properties to separate proteins.
 First, proteins may be purified according to their isoelectric points by
running them through a pH graded gel or an ion exchange column.
 Second, proteins can be separated according to their size or molecular
weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl
sulfate-polyacrylamide gel electrophoresis) analysis
 Thirdly, proteins may be separated by polarity/hydrophobicity via high
performance liquid chromatography or reversed-phase chromatography.

8.  Extraction
 Depending on the source, the protein has to be brought
into solution by breaking the tissue or cells containing it.
 There are several methods to achieve this:
 Grinding cells with sand or any abrasive
 Alternate freezing and thawing
 Placing cells in hypotonic soln.
 Ultrasonification/Enzymic treatment
 filtration

9. CENTRIFUGATION

10. CENTRIFUGATION
• Principal:
• Centrifugation imposes high centrifugal force on suspended particales
or even molecules in solution, and causes separation of such matter
on the basis of differences in molecular weight /density.
• On subjecting to ultracentrifugation,the lighter partical come to the
surface( floating),while heavier particles sink to the bottom of the
tube(sedimentation) and expressed as floating coefficient(sf) and
sedimentation coefficient(S) by svedberg .

11. Centrifugation
Centrifugation is a process which involves the use of the Centrifugal
Force for the sedimentation of heterogeneous mixture with a centrifuge
machine.
• Types
Based on RPM
 Micro centrifuges
 High-speed centrifuges
 Ultracentrifuges
Based on Process
 Differential centrifugation
 Equilibrium density-
gradient centrifugation

12. Types of Centrifuge based on RPM
• Micro centrifuges12,000–13,000 rpm
• High Speed Centrifuges  around 30,000 rpm
• Ultracentrifuges  excess of 70,000 rpm

13. Types of Centrifuge based on RPM
 Differential centrifugation
 Equilibrium density-gradient centrifugation

14. Differential Centrifugation

15. CLINICAL USES OF CENTRIFUGATION

16. ULTRAFILTRATION
• It is process of filtration under pressure for smaller particles to
expedite filteration.
• Special filters made of unglazed porclain are used.

17.  In bulk protein purification, a common first step to isolate
proteins is precipitation with ammonium sulfate (NH4)2SO4.
 This is performed by adding increasing amounts of ammonium
sulfate and collecting the different fractions of precipitate
protein. Ammonium sulphate can be removed by dialysis.
 Ammonium sulphate removes charges n hydroaic shell on the
protein molecules,the hydrophobic groups get exposed to the
atmosphere and it attracts other protein hydrophobic groups and
gets aggregated. Protein precipitated will be large enough to be
visible.
 One advantage of this method is that it can be performed
inexpensively with very large volumes.

18. Precipitation of proteins

19. DIALYSIS
• Passing protein soln. through semipermeable membrane,which
allows molecule to pass through according to size of the pores and
size of the particular protein molecule.
• Basis of artificial kidney
• Hemodialysis/Peritoneal dialysis

20. Chromatography
• Chroma means colour,graphy means write/measure
• Initially used to separate colour compounds, pigments from plant leaf
by Russian scientist, Mikhail in 1906.
• It is Misnomer,no longer limited to separation of colour compounds.

21. Chromatography Principle -
•
•
•
Very useful tech. by which extremely small amounts
of proteins and amino acids can be detected.
Chromatographic methods involve passing a
solution containing substances to be separated
(the mobile phase) through a medium,porous solid
matrix through which the sample percolates (the
immobile phase).
The interaction b/w the mobile and stationary
phases resuls in separation of the compound from
the mixture.
These interactions include physicochemical
principles such as Adsorbtion,partition,ion-
exchange,molecular sieving,affinity etc.

22. Chromatography
• The important methods of chromatography classified
depending upon nature of stationary phase are:
• Paper chromatography
• Thin layer chromatograpgy
• Column chromatograpgy

25. Paper chromatography
• Paper chromatography is an analytical method used to separate colored
chemicals or substances or aminoacids. It is primarily used as a teaching tool,
having been replaced by other chromatography methods, such as thin-layer
chromatography
• The protein is 1st hydrolysed by proteolytic enzymes/acid,
• Applied on one end a special filter paper sheet and dried,
• This end of paper is dipped into a special solvent,kept in vertical position,
• The capillary action forces solvent to move up,taking amino acids along with it
thus separating different amino acids.
• Paper is dried and stained with ninhydrin.Each a a has different resolution
front(Rf) value(dist. Traveled by aa/dist.traveled by the solvent).
• Rf values of U.K. aa can be compared with known aa in control tests.

26. PAPER CHROMATOGRAPGY

27. THIN LAYER CHROMATOGRAPGY
• Now preferred to paper chromatography.
• Thin layers of adsorbents(Alumina,silica gel etc.) are spread on glass
plats and dried used in the same way as the paper.

28. THIN LAYER CHROMATOGRAPGY

29.  Usually a protein purification protocol
contains one or more chromatographic steps.
 The basic procedure in column chromatography is to flow
the solution containing the protein through a column
packed with various materials.
 Usually proteins are detected as they are coming off the
column by their absorbance at 280 nm.

31. COLUMN CHROMATOGRAPHY
(Methods based upon interaction b/w mobile and stationary phase)
a-Gel Filtration Chromatography
b-Ion-Exchange Resins Chromatography
c- Affinity Chromatography
d-Adsorbtion chromatography
e-Gas liquid chromatography
f-HPLC (High Performance Liquid Chromatography)

32. Gel filtration (GF)
GF is simple to use and allows separation of
substances with differences in molecular size,
under mild conditions.
GF is a non-binding method

33. Gel Filtration Chromatography

34. Chromatofocusing (CF)
Chromatofocusing separates proteins according to
differences in their isoelectric point (pI).
It is a powerful method and can resolve very small
differences in pI
A pH gradient is generated on the column as buffer
and chromatography medium interact.

35. Anion exchange resins
(positive charge
e.g.NH2/NR3)
separate negatively
charged compounds,
cation exchange resins
(negative charge
e.g.COOH) separate
positively charged
molecules

36. Ion exchange chromatography (IEX)
 IEX separates proteins with differences in surface charge to give high-
resolution separation with high sample loading capacity.
 The separation is based on the reversible interaction between a charged
protein and an oppositely charged chromatography medium.
 Target proteins are concentrated during binding and collected in a purified,
concentrated form.

37. Affinity chromatography is a method of separating biochemical
mixtures based on a highly specific interaction such as that between
antigen and antibody, enzyme and substrate, or receptor and ligand.
Affinity Chromatography (AC)

39.  Resins used in the column are
amphiphiles with both
hydrophobic and hydrophilic
regions.
 The hydrophobic part of the
resin attracts hydrophobic
region on the proteins.
 The greater the hydrophobic
region on the protein the
stronger the attraction between
the gel and that particular
protein.

40. GAS LIQUID CHROMATOGRAPGY

41.  high pressure to drive the solutes
through the column faster.
 diffusion is limited and the
resolution is improved.
 The most common form is "reversed
phase" hplc, where the column
material is hydrophobic. The proteins
are eluted by a gradient of increasing
amounts of an organic solvent, such as
acetonitrile. The proteins elute
according to their hydrophobicity. After
purification by HPLC the protein is in a
solution that only contains volatile

42. HPLC

43. CLINICAL USES OF CHROMATOGRAPGY
• Chemical industry to detect impurities,like
pesticides,insecticides(DDT) in ground water,n PCB’s(polychlorinated
biphenyles in fish)- are removed with TLC.
• Used to prepare vitamins,preservatives,protein and amino acids.
• In pharmacy,chpromatography become crucial to analyze exact chiral
compounds and prepare large amount of pure materials.
• Used to isolate any amount ranging b/w m.gram to tons.
• To separate toxins from drinking water- testing tool by Govt.
• Forensic use in DNA,RNA fingerprinting, N fiber analysis.
• To detect Alcohol in blood or drugs.

44. ELECTROPHORESIS
• Means migration in an electric field
• Principle:The velocity of migration of each molecule in an electric
field is dependent upon the net charge on the molecule,strength of
the electric field and is inversely proportional to the molecular
weight.

45. • A typical electrophoresis apparatus is shown schematically
The porous support is hydrated and placed between the two chambers
containing a suitable buffer.
Sample is applied (in microlitres) on the support on the cathode end and the
components are allowed to move from cathode to anode under the influence of
direct current.
At the end of the run, the support is removed and the position of the molecules on
the support is ‘fixed’ with a fixative to prevent simple diffusion.
• The separated components are then stained to visualise them.
• The bands can be quantitated (by elution or by scanning with a densitometer)
as the uptake of the dye is directly proportional to the concentration of the
molecule in each band.

47. ELECTROPHORESIS
 Electrophoresis in gels are efficient than in solutions.
 Proteins are exposed to the ionic detergent SDS.
 The pores in a polyacrylamide gel are quite small.
 The rate of movement is influenced by
 the gel’s pore size
 the strength of the electric field.

48. n

 Gel electrophoresis is a common laboratory
technique that can be used both as preparative and
analytical method.
 The principle of electrophoresis relies on the movement
of a charged ion in an electric field. In these conditions,
the proteins are unfolded and coated with negatively
charged detergent molecules. The proteins in SDS-PAGE
are separated o the sole basis of their size.
In analytical methods, the protein migrates as
bands based on size. Each band can be detected using
stains such as Coomassie blue dye or silver stain.
Preparative methods to purify large amounts of protein
require the extraction of the protein from the
electrophoretic gel. This extraction may involve excision
of the gel containing a band, or eluting the band
directly off the gel as it runs off the end of the gel.

49. SDS-Polyacrylamide Gel Electrophoresis

50. SDS PAGE
• Separates Proteins based on their sizes
• Separates Proteins from DNA, Lipids and Polysaccharides
A B C D E F G

51. Clinical uses of electrophoresis
• Serum protein electrophoresis
• Lipoprotein analysis
• Diagnosis of hemoglobinopathies n HbA1c
• Determination of serum protein phenotypes e.g. alpha,1-antitripsin
defficiency MM.
• Genotyping of proteins e.g. ApoE analysis for Alzheimers disease.
• Cerebrospinal fluid analysis.
• Urin analysis(GN’s)

52. Enzyme-Linked Immuno-Sorbent
Assay(ELISA).
• It is used to detect non-catalytic protein in extremely small amount.
• It employs an immunosorbent(serumAg/Ab bound to a solid support
e.g.a plastic micrititre plate) and an enzyme-labeled
immunoreactant(Ag/ Ab specific for immunosorbent, covalently
linked to reporter enzyme).The immunosorbent binds the
immunoreactant depending upon the amount of the latter.
• Afterwards the amount of the bound immunoreactant is found out by
determining the enzyme linked to it by adding the appropriate
substrate,from which serum or sample level of the immunosorbent is
determined.

53. Proteomics
• Proteomics is the science of protein
expression of all the proteins made by a
cell
• Proteome pertain to all proteins being
made according to the transcriptome
(RNA profile).
• It is often visualized by a system
interaction map as seen in the
proteogram.

54. Procedures of the Proteomics
• Commonly used procedures by
Proteomics are:
• Mass Spectrophotometry – detects exact
mass of small peptides (molecular weight).
• X-ray Crystallography – determines 3D
shape of molecules mathematically
• NMR Spectroscopy – magnetic signal
indicates distances between atoms

55. Spectrophotometry
• Measurement of light absorbtion or transmission
• U.K.compounds can be identified by their characteristic absorbtion
spectra in ultraviolet or infrared regions of the electromagnetic
spectrum.
• Concentratio o U.K. compounds in soln. may be determined by
measuring light absorbtion at one or more wavelengths by formula:
• Conc.of U.K.=Absorbance of U.K./Absorbance of std.x Conc. Of std

56. Present Scenario
 Protein purification is now performed in scales from
micrograms and milligrams in research laboratories to
kilograms and tones in industrial settings.
 The efficiency gained by the generic purification approaches
based on affinity tagging of the target protein has
revolutionized protein purification
 The challenges in protein purification that still remain make it
worthwhile to gain solid knowledge about protein purification so that
the available methods can be selected and applied in an optimal way.
 Some proteins may be very challenging to purify in an active and
stable form.

57. References
 Ion Exchange Chromatography & Chromatofocusing Principles and Methods- Online GE Handbook.
 Methods for Protein Analysis- MITTv
 Purifying Challenging Proteins-Principles and Methods: GE Healthcare Handbook
 Ion Exchange Chromatography- Theory and Principles,
https://www.youtube.com/watch?v=A8lTfhWdAwE
 Cell Fractionalism
,http://www.sumanasinc.com/webcontent/animations/content/cell_fractionation.swf

58. End of
Presentation
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