1. What elutes out of gel filtration/size exclusion chromatography first?
2. How do you collect proteins of interest from natural binding affinity chromatography? (2)
3. What affects how protein will move in electric field through SDS Page? (3)
4. What does SDS confer on all proteins? (2) How? What does it separate everything based on?
5. How are proteins later visualized?
6. Do smaller or larger proteins move farther?
7. What does SDS leave intact?
- 1. Large proteins
- 2. Excess salt/excess free ligand solution
3. Size, shape, and charge
4. Same shape (linear) & charge:mass ratio.
Shape - SDS breaks all noncovalent bonds, denaturing protein into its linear form.
Charge - SDS is highly negative, binds all around protein, making it super negative. Separates everything based on MW.
5. Coommassie blue
7. Disulfide bonds (b/c they're covalent)