Affinity Chromatography Flashcards
Affinity chromatography
Uses a ligand bound to the solid phase
This ligand can be designed so that it has high specificity for the protein of interest
Because binding to the ligand is reversible, a competitive inhibitor is then used to elute the protein
Types of affinity chromatography
- Ligand/inhibitor
- Poly-histidine tags
- GST tags
- Immunoaffinity chromatography
Ligand/inhibitor chromatography
In theory the ligand should have affinity for only one protein
When the mixture is added to the column, only the protein of interest should bind
The other compounds can be removed with a salt wash
The compound of interest is then eluted using a competitive inhibitor
Poly-histidine tag chromatography
Can be used when a suitable ligand is not available
Molecular biology techniques are used to add a histidine tail to the protein of interest
The matrix is then set up containing nickel ions which have high affinity for histidine
Imidazole is then used like a competitive inhibitor to elute the protein
GST tags
Rarely used nowadays
A fusion protein is synthesised; the protein is coupled with GST
A matrix is then set up containing GSH, which binds to GST
However, the original protein is now not present, although the GST tag may be able to be removed
Hydrophobic interaction chromatography
Uses the hydrophobicity of the protein of interest to bind to the matrix
A hydrophobic matrix binds to hydrophobic areas of the protein
Elution is carried out by decreasing the salt concentration
Chromatofocusing
Proteins are eluted in order of their isoelectric points
The overall charge of the protein binds it to the matrix
A pH gradient is then used; as a zwitterion forms the protein can no longer bind to the matrix and so is eluted
Post-chromatography processing
- desalting/ buffer exchange
- concentration
- yield/analysis
