2D Protein Electrophoresis

Theoretical Background/Context

• Within any cell, the genome is constant, but the proteome varies and is dynamic. 
• The proteome is the whole set of proteins produced by an organism in a cell. 
• It varies with time and divergent statuses or stresses subjected to that cell or organism.
• Isoelectric Focusing (IEF) [1st dimension] is a technique for separating molecules according to their isoelectric point (pI). 
• A protein present in a pH region below its pI will carry a positive charge; therefore it migrates towards the cathode (negatively charged electrode). 
• While it migrates through an increasing pH gradient, the protein's charge decreases until it reaches the pH region that corresponds to its pI. 
• This is where the protein has zero net charges so it stops migration. 
• This is simply how proteins focus into sharp bands. 
• The second dimension depends on SDS-PAGE (SDS-polyacrylamide gel electrophoresis), which is an electrophoretic method for separating polypeptides according to their molecular weights (Mr).
• This way proteins carrying the same pI can be further separated in a second dimension according to their Mr. 
• The technique is performed in polyacrylamide gels containing sodium dodecyl sulfate (SDS). The intrinsic electrical charge of the sample proteins is not a factor in the separation due to the presence of SDS in the sample and the gel. 
• When proteins are treated with both SDS and a reducing agent, the degree of electrophoretic separation within a polyacrylamide gel depends largely on the molecular weight of the protein.
• In this way, a mixture of thousands of different proteins can be separated using 2D protein electrophoresis and the relative amount of each protein can be determined.

Principle of 2D Electrophoresis

• Cellular proteome can be simultaneously separated using 2D Protein Electrophoresis. 

This technique separates proteins in two steps, according to two independent properties:

1. First-dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI).
2. Second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (MW):

1. Preparing the second-dimension gel for 2D protein electrophoresis.
2. Equilibrating the IPG strip(s) in SDS buffer. 
3. Placing the equilibrated IPG strip on the SDS gel.
4. Electrophoresis and staining.