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affinity chromatography.pdf
Affinity chromatography is a technique that uses the specific binding between a ligand and target molecule to separate a target from a complex sample. It works by immobilizing a ligand with specific binding affinity for the target molecule on an insoluble support. The sample is passed through the column, and the target molecule binds reversibly to the ligand. The bound target can then be eluted selectively under appropriate conditions. Key components of an affinity gel include the matrix, spacer, and ligand. Researchers can also design their own affinity gels by raising antibodies or choosing a suitable ligand to isolate a protein of interest, such as using an agarose gel with immobilized antibodies to purify the Rubisco enzyme from plant extracts.
affinity chromatography.pdf
- 1. Affinity Chromatography Concept Design DIY Application Source ofprotein Extraction Separation Purity & characterization Ref: Scopes RK Protein Purification. Principles and Practice (3rd Ed) Chapter 7
- 2. Affinity Chromatography Concept Adsorption chromatography Adsorptionis 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.
- 4. Type of bindingspecificity
- 5. Design Components of affinitychromatography Matrix Spacer Ligand Molecule of interest
- 6. Commercially available affinityabsorbents
- 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 withfunctional groups for ligand binding
- 10. Is spacer needed?Yes and No, depending on situation
- 11. Sephacryl S200 chromatography Con Aaffinity chromatography Elute with methyl α- D mannoside
- 13. DIY Affinity gel forisolating Rubisco Agarose CNBr antibody Rubisco CNBr activation is widely used for attachment of proteins through ε-lysine group. Cyanogen bromide activated agarose is available commercially.
- 14. Rubisco affinity gel Whatyou 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 antibodyproduction 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 Rubiscoby affinity chromatography Source: Yeoh HH, Stone NE and Watson L (1981) Biochem Syst Ecol 9, 307-312
- 17. Summary Affinity Chromatography Understand theprinciple 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 Saltprecipitation Gel filtration Ion exchange Dialysis step Chromatofocusing Hydrophobic Interaction Chromatography Affinity Chromatography