experiment 8: multi-column purification of rGFP

Question 1

what are the underlying principles of separation used in gel filtration chromatography? ion-exchange chromatography?

Answer 1

gel filtration: separates by size or molecular weight

ion-exchange: separates based on charge-charge interactions (anion or cation exchanger)

Question 2

what happens to the protein amount, protein purity, and/or specific activity of a purification fraction if one of the three is changed?

Answer 2

as purity increased along with the specific activity of the fraction, however the total protein amount and activity should decrease

Question 3

what is the difference between a linear gradient and a step gradient and how proteins might separate differently using one or the other gradient?

Answer 3

linear: concentration of salt used to elute sample gradually increases over time
step: concentration of salt used to elute sample increases each step

with a linear gradient, you can resolve the difference in each peak and have more distinct peaks. with a step gradient, different proteins might show up as one broad peak, and not as defined peaks.

Question 4

what changes can optimize protein separation?

Answer 4

high vs low bed volumes
tall/thin vs short/fat column
slow vs fast flow rate

Question 5

be able to predict the effect of varying the salt concentration to elute proteins off of both strong and weak exchangers

Answer 5

weak exchanger: nature of exchanger changes over time and only half of the resin molecules are charged.
lower salt concentrations are able to elute off proteins.

strong exchanger: nature remains constant over time and all of the resin molecules changed.
higher salt concentrations are necessary to elute off highly charged proteins

Question 6

be able to determine the effectiveness of a multi-column procedure given a purification table

Answer 6

activity should decrease
total amount of protein should decrease
purity should increase
specific activity should increase