Question

a. Which spot corresponds to the protein with the largest molecular weight?

isoelectric focusing electrophoresis pH 5 pH 9

b. When pH = 7, what order would the following peptides elute from a column filled with cation exchange resin? Size exclusion column?

Protein 1 – 400 kDa;
10%A 5%E, 5%F, 10% G, 5% H, 10%I, 10%K, 10%L 10%R, 5%P, 5%S, 10%W, 5%Y

Protein 2: 40 kDa
5%A, 5%C, 5%D, 5%E, 5%F, 10%G, 5%I, 10%K, 5%L, 5%M, 5%N, 10%P, 5%R, 5%S, 5%T, 5%V, 5%W,

Protein 3 – 175 kDa
10% A, 5% C, 10% D, 10% E, 5% F, 5% G, 5% H, 5% L, 5% M, 5% N, 5% P, 5% Q, 5% R, 5% S, 5% T, 5% V, 5% Y

--I think the order is 3,2,1 for elute, but what about size exclusion?

Answer 1

1. Answer Molecule B.

In iso electric focusing the molecule move until it reach the PI value of the protein. Then the column kept in SDS page the small molecule move fast and the larger molecule move very slowly. So, the molecule B is the largest one it move slowly in the PAGE.

2. size exclusion - The order 1, 3, 2

In size exclusion chromatography the larger molecule elute first it not enter into the pores so it directly elutes first. The small molecule enter into the pores and finally elutes out.