Purification and Analysis of Proteins

What are the two general methods for Purification and Analysis of Proteins

Chromatography and electrophoresis.

What are the three types of chromatography?

1. Gel filtration

2. Affinity

3. Ion exchange

What is gel filtration chromatography also known as?

Size exclusion chromatography

How does gel filtration chromatography work?

It separates proteins based on their size, allowing smaller molecules to enter the pores of the gel beads while larger molecules elute first.

What elutes first in gel filtration chromatography? Why?

Larger molecules elute first because they cannot enter the pores of the gel beads.

What are the two methods of detecting proteins in gel filtration chromatography?

1. Spectrophotometer absorbance at 280nm

2. Colorimetric Assays

What does spectrophotometer absorbance at 280nm detect?

It detects the presence of aromatic amino acids, primarily tryptophan and tyrosine, in proteins.

What are the two types of colorimetric assays?

1. Bradford Assay

2. Lowry Assay

How does the Bradford assay work?

The binding of dye to proteins, resulting in a color change that can be measured spectrophotometrically.

The intensity of the color is proportional to the protein concentration.

How does the Lowry Assay work?

It measures the reduction of copper ions by proteins in an alkaline solution, leading to a color change that can be quantified spectrophotometrically.

This change correlates with protein concentration.

How does affinity chromatography work?

Separates based on binding partner. The stationary phase includes specific ligands that interact with the target protein.

This allows for the target protein to bind while unbound proteins are washed away, after which the target can be eluted.

In affinity chromatography, what are 4 ways that you can elute the protein of interest?

1. Add extra free ligand

2. Change the pH

3. High salt concentration

4. Denaturing solution

What is ion exchange chromatography? How does it work?

Separation based on charge

There is a positive or negative charge on the column. Proteins with opposite charges bind to the resin, allowing unbound proteins to be washed away before elution.

(anion exchangers —> positive charge on the column)

(cation exchangers —> negative charge on the column)

What is electrophoresis?

Separation of biomolecules (including proteins) by migration in an electric field

What are the 3 types of electrophoresis?

1. Native PAGE (polyacrylamide gel)

2. SDS PAGE

3. Reducing SDS PAGE

What does Native PAGE do?

Separates based on size and shape

What does SDS PAGE do? How does it work?

Separates proteins based on molecular weight (mass)

It denatures the proteins with SDS, which gives all proteins a uniform negative charge.

What is SDS?

A detergent that denatures proteins and gives a negative charge.

What is Reducing SDS PAGE? How does it work?

A variation of SDS PAGE that breaks disulfide bonds in proteins using reducing agents

This allows for more complete denaturation and accurate molecular weight determination.