Chapter 3 (Section 3.3: Working with Proteins)

What differences does protein separation rely on

• charge

• size

• affinity for a ligand

• solubility

• hydrophobicity

• thermal stability

The protein is often able to remain fully folded during

purification. Why is this important?

If a protein unfolds (denatures) during purification, it usually loses its ability to function.

What are the steps to purifying proteins

1. Break open tissue or mircocial cells (crude extract)

2. Separate proteins into fractions based on size and charge (fractionation)

3. Use semipermeable membrane to separate proteins from small solutes (dialysis)

What is crude extract

Releases protein into the solution

What is “Salting out”

lower solubility of proteins in salt to selectively precipitate proteins

Column Chromatography

• Allows separation of proteins over a solid phase using liquid phase to move proteins

• Proteins with lower affinity for the solid phase will leave the columns first

• Takes advantage of charge, size, and binding affinity

Ion Exchange Column Chromatography

• Separation by charge

• Proteins move through the columns based on charge at the pH used

• With cation exchangers, proteins with a negative charge move faster and elute earlier

• Contains cation exchangers

Size Exclusion Column Chromatography

• Separated based on size

• Larger molecules pass more freely which appear in the earlier fractions

• Contains cross-linking polymer

Binding Affinity Column Chromatography

• Separated based on binding affinity

• Protein of interest is eluted by ligand solution and unwanted proteins are wash through column

• Containing a polymer-bound ligand specific for protein of interest

Liquid Chromatography

Uses high pressure pumps to move proteins down a very tight packed column which improves resolution

What are the benefits to Gel Electrophoresis

Visualize and characterize purified proteins

Gel electrophorisis can be used to estimate:

• Number of different proteins in a mix

• degree of purity

• isoelectric point

• Approximate weight

Why is separation on an analytical scale commonly done by gel electrophoresis

• An electric field moves proteins based on charge

• The gel hinders the mobility based on size and shape

• The gel is made of polyacrylamide gel electrophoresis (PAGE)

SDS PAGE

• Separates based on molecular weight and can calculate weight

• SDS micelles bind to proteins and facilitate protein unfolding

• Small proteins move faster

• Native shape does not matter

• Uniformly gives all proteins a negative charge

2D Electrophorisis

Uses SDS-PAGE and Isoelectric Focusing which allows us to see a clear resolution of the proteins

What is the chemical used in SDS PAGE

Sodium dodecyl sulfate is a detergent

What is the activity of the protein

The function

What is the specific activty of a protein

The ratio of activity to total protein concentration

Why is higher specific activity in a protein more favorable

it indicates higher purity and enrichment of the target enzyme relative to total protein mass