Protein purification and determination of specific activity

Question 1

Size exclusion/gel filtration chromatography

Answer 1

-separates by size (smaller prots take longer)
-bead column
-prots don’t bind to matrix

Question 2

Ion exchange chromatography

Answer 2

-separates by charge
-charged aa residues interact with charged surface of matrix

either anion (+ve matrix binds -ve molecules) or cation (-ve matrix binds +ve molecules) exchange

-change pH to elute protein

Question 3

Anion exchange vs cation exchange chromatography

Answer 3

anion exchange column is made up of a +ve matrix so binds -ve molecules
cation exchange column is made up of a -ve matrix so binds +ve molecules

Question 4

Isoelectric point (pI)

Answer 4

the pH where a molecule (eg. protein) has no net charge
charge = 0

-use pI to determine whether to use anion or cation exchange column; what conditions and buffer to use; how to elute the protein

Question 5

Hydrophobic interaction chromatography

Answer 5

-hydrophobic residues on prot surface interact with hydrophobic matrix surface
-requires high salt conc (so that no water is present so residues def interact with matrix instead of water)

-salt conc decreased to elute prot

Question 6

Affinity chromatography

Answer 6

-specific interactions between protein and matrix
-use tagged proteins (tag interacts with matrix) or pseudo-affinity (ligand bound to matrix v. sim to native ligand/substrate of the prot)

Question 7

Dye chromatography

Answer 7

-type of pseudo-affinity chromatography
-ligand bound to matrix is made from synthetic polycyclic dyes which have v. sim struct to cofactors like NAD(H) and NADP(H)
-used for purifying dehydrogenases and kinases

Question 8

Psuedo-affinity chromatography

Answer 8

-ligand attached to the matrix is v. sim to the structure of the native lig/substrate of the prot
eg. antibodies, dyes, etc

Question 9

Why does hydrophobic interaction chromatography require high salt concentrations?

Answer 9

ensures that no water is present so that residues interact with matrix instead of water (if water was there, they’d pick water over the hydrophobic matrix!)

N/B: afterwards do need to decrease saltiness to elute prot!