affinity chromatography.pdf

Affinity chromatography

1. Affinity Chromatography
Concept Design DIY Application
Source of protein
Extraction Separation
Purity &
characterization
Ref: Scopes RK Protein Purification.
Principles and Practice (3
rd
Ed) Chapter 7

2. Affinity Chromatography
Concept
Adsorption chromatography Adsorption is highly specific to binding substance (ligand) Process is reversible Binding substance is
immobilised to an insoluble
support (matrix)
Source of protein
Extraction Separation
Purity &
characterization

3. sample ligand
reversible
Specific
binding

4. Type of binding specificity

5. Design
Components of affinity chromatography
Matrix Spacer Ligand Molecule of
interest

6. Commercially available affinity absorbents

7. Properties of matrix
Quality: Important if you are developing your own
affinity absorbents
insoluble with suitable chemical group for ligand attachment must be stable during binding of sample be chemically inert towards sample have uniform structure for good flow rate
Types available
cellulose agarose, cross-linked agarose polyacrylamide porous glass/ceramics

8. Available matrix with functional groups for ligandbinding

10. Is spacer needed? Yes and No, depending on
situation

11. Sephacryl S200
chromatography
Con A affinity
chromatography
Elute with methyl α-
D mannoside

13. DIY
Affinity gel for isolating Rubisco
Agarose CNBr antibody Rubisco
CNBr activationis widely used for attachment of
proteins through ε-lysine group. Cyanogen bromide
activated agaroseis available commercially.

14. Rubisco affinity gel
What you need to do
Raise antibodies Isolation and
purification
Binding of antibodies to matrix Develop a protocol for binding and elution
Considerations
Antibodies
Amount of antibodies Choice of animal systems Protocol to raise antibodies If hapten is required
Preparing the gel
Which reagent to use for
binding of ligand to matrix
Is spacer arm needed?

15. Stages in antibody production
Antigen +
adjuvent
emulsifer
Animal system eg rabbit
~ 10 days, IgM
Repeat
injection of
same antigen
~ 3 days, gM and
IgG
Intial dosage
Measure titre
Purify IgG

16. Isolation of Rubisco by affinity chromatography
Source: Yeoh HH, Stone NE and Watson L (1981) Biochem Syst Ecol 9, 307-312

17. Summary
Affinity Chromatography
Understand the principle behind affinity
chromatography
Appreciate the concept Know the components of affinity gel Know how to design own affinity gel Able to interpret published information on affinity gel

18. Crude enzyme extract
Salt precipitation
Gel filtration
Ion exchange
Dialysis step
Chromatofocusing
Hydrophobic
Interaction
Chromatography
Affinity Chromatography