Question

A protein with a high percentage of lysine and arginine residues would be BEST purified and concentrated with which type of column? Explain

A. cation exchange

B. anion exchange

C. size exclusion chromotagraphy

D. affinity chromotagraphy

Answer 1

Answer (A) :- Cation-exchange chromatography:
2) negatively charged resin (negative charges on the surface); therefore, there is an exchange of cations
- generally bound to Na+ or K+

Answer (B) :- Anion-exchange chromatography:
3) positively charged resin (positive charges on the surface); therefore, there is an exchange of anions
- generally bound to Cl-

4) Exchange resin is bound to counterions
5) Mixture of proteins is loaded on the column and allowed to flow through
6) Proteins that have a net charge opposite to that of the exchanger stick to the column, exchanging places w/ the bound counterions
7) Proteins that have no net charge or same charge as the exchanger elute
8) After unwanted proteins are eluted, eluent is changed either to a buffer that has a pH that removes the charge on the bound proteins or to one w/ a higher salt concentration
9) Higher salt concentration will outcompete the bound proteins for the limited binding space on the column
10) Desired molecules are eluted

Answer (C) :- Size-exclusion (or gel filtration) chromatography is a form of column chromatography

Advantages:
1) proteins are separated by size
2) estimate molecular weight by comparing the sample w/ a set of standards

Stationary phase composed of cross-linked gel particles:
1) particles are in bead form
2) consists of one of two kinds of polymers; a carbohydrate polymer (dextran or agarose) or polyacrylamide

How it works:
1) Cross-linked structure of these polymers produce pores in the material
2) Extent of cross-linking can be controlled to produced desired pore size
3) Smaller molecules enter the pores and are delayed in elution time (only after escaping the pores)
4) Larger molecules do not enter and elute from column before smaller ones

Answer (D) :- Form of column chromatography

Advantages:
1) Uses specific binding properties of molecules/proteins
2) Produces very pure proteins

How it works:
1) Stationary phase has a polymer that can be covalently linked to a compound called a ligand that specifically binds to the desired protein
2) After the unwanted proteins are eluted, more ligand or salt is added
3) Salt weakens the binding of protein to the ligand and extra free ligand competes with the column ligand in protein binding
-a change of pH or ionic strength will disrupt the ligand-protein interaction-

Use this after getting partially pure protein to get pure protein

Problems:
1) ligand doesn't bind properly to polymer
2) ligand binds too strongly; can't remove
3) expensive