Chapter 21: Protein Purification

SEC separation basis is based on what?

molecular size

Elution order of SEC

large proteins first

Ion exchange separation basis is based on what?

charge

What is the elution order of ion exchange?

weak binding first

Affinity separation basis is based on what?

specific binding

What is the elution order of affinity

non-binding first

Salting out (precipitating)

Ammonium sulfate precipitation — removes unwanted proteins, concentrates sample

Making a substantial change to the salt concentration of a solution affect protein solubility, causing some proteins to precipitate

At different concentrations of salt, different proteins become insoluble and precipitate

Dialysis

Removes small molecules through semipermeable membrane, buffer exchange

Process in which a protein solution can be either desalted or changed from one buffer to another wihtout precipitationg the protein of interest

As purification progresses, what happens when specific activity increases (units/mg)

protein becomes purer

As purification progresses, what happens when the total yield decreases

some protein lost at each step

As purfication progresses and fold purification increases

measure of enrichment

salting in (solubilizing)

Addition of certain salts (low conc. of salt) such as guanidinium chloride increases the solubility