Protein Purification Flashcards
What is the purpose of protein purification?
To separate proteins based on charge, size, affinity for a ligand, solubility, hydrophobicity, or thermal stability
What are the methods for protein purification?
Chromatography, electrophoresis, isoelectric focusing, and 2D electrophoresis
What is chromatography used for?
Preparative separation because the proton can often remind fully folded with this method
What are the types of chromatography?
Column chromatography, ion exchange chromatography, size exclusion chromatography, and affinity chromatography
Column chromatography:
Separation of a mixture into solid and liquid phase with a porous membrane, proteins with a lower affinity for solid phase are removed first
Ion exchange chromatography:
A crude extract is placed on top of a sample, proteins with an affinity for its properties (specifically charge) will rise towards it and those without will sink and be removed, used to sort by charge
Size exclusion chromatography:
Columns contain cross-linked polymers which proteins have to migrate through, larger PRO will migrate first because they won’t fit through all the smaller pathways, smaller will take longer because they have more pathways to cover
Affinity chromatography:
A ligand is added to the column, proteins with an affinity for it will bind to it and those without will not, either the ligand will be removed with the bound proteins or the unbound proteins will be removed
What is electrophoresis/how does it function?
Uses an electrical field to pull proteins through a gel, this separates them based on shape and size (thus molecular weight)
What is the role of SDS-PAGE in electrophoresis?
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel) is used to denature the proteins to give them a uniform shape and gives them a uniform charge so they all have an equal attraction towards the electrical field, this gives better/more specific results
What is isoelectric focusing and how does it function?
Used to determine the pI of proteins
Used a gel strip with a pH gradient, the electric field pulls proteins through the strip and they stop based on their pI
What is 2D electrophoresis?
Combination of SDS-PAGE and isoelectric focusing, used to map proteins by molecular weight and pH in one test
How would you yield the most specific results for protein purification?
Put the protein sample in question through all the tests to find the most specific results
