Wize University Biology Textbook > Lab Techniques for Biology

Protein Purification

Protein Purification Techniques

Centrifugation: Size Separation

Proteins vary in MASS but not much in DENSITY.

Differential Centrifugation - First step in most protein purification because it is used to separate soluble proteins from cell organelles. The organelles (heavy) form a pellet at the bottom, and the less dense soluble proteins can be collected from the supernatant.

Rate-Zonal Centrifugation - Proteins are separated through a density gradient (usually a sucrose gradient) and form discrete "zones/fractions" which can be isolated.
Note: Rate-zonal centrifugation is good for separating proteins from one another but not reliable for determining the mass of these proteins.

Photo by Jolozaga / CC BY

Liquid Chromatography: Size, Electric Charge, and Affinity Separation

Gel-Filtration Chromatography - separates proteins based on mass.
A column is filled with porous beads. Small proteins get trapped in the beads more easily and move more slowly down the column while large proteins move quickly through them.

Photo by Potcherboy / CC BY

Ion-Exchange Chromatography - separates proteins based on charge.
A column is filled with charged beads (either negatively (anion) or positively charged (cation)).
If the beads are positively charged, acidic (negatively charged) proteins will stick to them.
If the beads are negatively charged, basic (positively charged) proteins will stick to them.
A highly concentrated salt solution is then used to elute the proteins off of the beads (because the salt ions are more negatively charged than the proteins).
Proteins with low/neutral charges are eluted first and more highly charged proteins are eluted last.

Affinity Chromatography - separates proteins based on affinity for certain antibodies.
A column is filled with beads attached to a specific ligand or antibody.
Only proteins that bind to that specific ligand or antibody will get trapped on the column.

Practice: Protein Purification

You have a mixture of three proteins that have the same net charge but are all different sizes. Which of the following is the best method to separate the proteins for further analysis?

A) Two Dimensional Gel Electrophoresis

B) Gel-filtration Chromatography

C) Ion-exchange Chromatography

D) Affinity Chromatography

E) Immunoprecipitation

I don't know

Extra Practice

Protein analysis

A asymmetrical multi-subunit protein consists of all three secondary structures and disulfide bonds. It has a total molecular weight of 56 kDa. Answer the following questions about this protein.

Protein Separation Methods

Which of these options would not be a possible step in a protein purification protocol?

List the stages of a typical protein purification workflow in order.

Which of the following methods is NOT an effective way to purify or detect a protein based on its size?

If centrifugation was used to separate key cellular components in a viscous sugar solution, what layer would you expect to find the following components:

If differential centrifugation was used to separate cell lysate solution, in what order do you expect the following to be extracted?

1. Nuclei

2. Endosomes