Methods for protein separation and purification

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What is Centrifugation ?

usually the 1st step-- spinning a mixture of proteins in a centrifuge causes them to move to the bottom of the tube at different rates DEPENDING ON MASS AND DENSITY

What are the 2 main types of centrifugation?

1. differential centrifugation
2. rate zonal centrifugation

What is differential centrifugation?

Separates proteins based on DENSITY AND SIZE; spin at specific speeds; form pellets

What is rate zonal centrifugation?

based on MASS AND SHAPE; proteins spun for a shorter, fixed period of time; separate in discrete bands based on size.

Electrophoresis

separating molecules in a mixture by adding the mixture to a semi-solid gel and applying an electric current through a polyacrylamide gel.

-separate based on charge,mass,3-D shape
[small molecules move faster]

SDS-PAGE

type of electrophoresis that uses the negatively charged detergent SDS and heat to denature proteins prior to adding them to the gel.

- SDS coats all proteins with negative charge/removes all 2nd,3,4 structures
[proteins separate on SIZE only]

Liquid chromatography

mixture of proteins is placed on top of a tightly packed column of spherical beads--proteins move through the beads-- proteins separate based in differences in mass, charge, binding affinity depending on type of bead used--fractions of the liquid flow are collected-- different proteins should be in different fractions

What are the three major types of Liquid chromatography and how do they differ?

1. gel filtration chromatography
2. Ion-exchange chromatography
3. Affinity chromatography

gel filtration chromatography

-based on SIZE;
-beads have depressions over surface--LARGER PROTEINS COME OUT 1ST BECAUSE they don't get stuck in the bead depression

Ion-exchange chromatography

-based on CHARGE
- + or - beads are used.. positively-charged beads will bind to any proteins that are negatively charged (vice versa).. proteins of the same charge of the beads just go through the column

bound proteins are removed form the beads by adding NaCl (salt disrupts ionic interactions)
(the stronger the attraction, the more salt required)

Affinity chromatography

-based on their attraction to a specific ligand molecule
-ligand molecules that bind to the protein that you are trying to purify will be chemically attached to the beads--only those proteins with affinity for the bound molecule will stick to the beads
MOST SPECIFIC OF THE LIQUID CHROMATOGRAPHY TECHNIQUES

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