Protein Chromatography Flashcards
How does precipitation of proteins work
Aas form bonds with water. Increasing salt displaces water. Hydrophobic regions are now exposed causing proteins to coagulate and precipitate.
Reasons to purify a protein
Biochemical assays Characterize activity, structure, function Therapeutic apps Biotechnology Raise Abs for downstream uses
Ability of salt to precipitate proteins called
Hofmeister series
Types of liquid chromatography
Affinity
Ion exchange
Hydrophobic interaction
Gel filtration
Affinity chromatography separates on the basis of
Reversible interaction with ligand.
Popular small affinity tags
His, FLAG, strep ll, s-peptide
Popular large affinity tags
MBP, GST, cellulose-binding domains, calmodulin binding peptide. His-patch thioredoxin
Ion exchange separates on basis of
Net surface charge (net charge vs pH titration curve)
What does amphoteric mean
Charge changes as pH changes
Hydrophobic interaction chromatography works on basis of
Surface hydrophobicity - high salt increases interaction with hydrophobic ligands
What is unique about gel filtration
Molecules do not bind the column. Therefore, buffer composition can suit downstream apps.
Can also contain any salt concentration/ cofactors/ metal ions
What is void volume
Total volume of column minus volume of beads
What is Kav
A partition coefficient related to the size of a molecule. Globular proteins have a linear relationship between K av and log Mr (exclusion limit)
What does an increase in number of purification step do
Decreases yield
