4. SEC

Question 1

What is the principle behind SEC?

Answer 1

Proteins are separated based on MW/size. Column has pores of a certain size that only proteins small enough can diffuse through, hence, they elute later. Larger proteins flow between the beads, and are eluted first, followed by intermediate MW, then proteins of lowest MW.

Question 2

What is the general flow of SEC?

Answer 2

1. Load sample
2. Sample flows through column containing beads of different pore sizes which is the stationary phase.
3. Smaller proteins diffuse through pores so they retain longer.
   Larger proteins flow between beads so they elute first.

Question 3

What is Vo, Vt, and Ve?

Answer 3

Vo = void volume = volume outside of beads = proteins that cannot enter pores.
Vt = total column volume including beads.
Ve = elution volume = volume required to elute protein at its peak of its elution profile.

Question 4

What are the 2 characteristics of the column matrix?

Answer 4

1. It is made of sephadex (cross linked dextran), agarose, silica, or polyacrylamide.
2. It should not adsorb proteins non-specifically to ensure proper separation.

Question 5

Using Vo and Vt, when do larger and smaller proteins elute?

Answer 5

Larger proteins elute at Vo. Smaller proteins elute after Vo and before Vt.

Question 6

How do you know when your POI has eluted?

Answer 6

By figuring out Vo which can be done using blue dextran protein. It is a high MW protein and will elute first which indicates Vo. Proteins that elute after are smaller.

Question 7

Explain what kind of peaks smaller proteins would have.

Answer 7

Their peaks would be closer to one another as their sizes are less different.

Question 8

What are the factors that affect resolution?

Answer 8

1. Flow rate: slower flow rate, better resolution.
2. Sample volume: smaller volume, better resolution.

Question 9

What are 3 applications of SEC and explain them.

Answer 9

1. Separating glycoproteins and non glycoproteins: sugar region of glycoproteins labelled with radioactivity which allows for glycoproteins to be detected.
2. Desalting: salt size smaller than pores while proteins are larger. Salt diffuses and retains longer in pores while proteins elute first.
3. Dialysis: salt size smaller than pores of membrane while proteins are larger. Salt diffuses out of membrane while proteins retain in membrane.

Question 10

How to determine MW of unknown protein using SEC?

Answer 10

By using known protein standards.
1. Determine Vo using blue dextran.
2. Determine Ve of protein standards.
3. Plot logMW against Ve/Vo.
4. Determine Ve of unknown protein.
5. Get its Ve/Vo and match to graph to find its logMW then determine MW from there.