lab procedures 9/19

Question 1

if the desired protein is a membrane protein…

Answer 1

the membrane must be solubilized to allow further fractionation

Question 2

what is salting in and out and what is usually used

Answer 2

salting in - proteins come out in solution

salting out - (NH4)2SO4

trichloroacetic acid or acetone - often used to precipiate proteins from a solution

Question 3

molecular exclusion chromatography

Answer 3

polymer beads with holes - small proteins go through channels and slower while larger move faster

Question 4

specific affinity chromatography

Answer 4

• purest sample
• stationary has ligand and protein may stick to ligand and move slower

Question 5

what is electrophoresis

Answer 5

using an electric field to separate proteins based on their electrophoretic mobility

Question 6

SDS-PAGE

Answer 6

• separate proteins based on size alone
• binds to hydrophobic protein parts and makes them unfold into the same shape
• can measure unknown weight next to known weights (log relationship)

Question 7

isoelectric focusing

Answer 7

(IEF) separates based on pI

Question 8

2D electrophoresis

Answer 8

• combination of IEF and SDS-PAGE
• top: big proteins
• left: acidic