2 Dimensional Protein Electrophoresis

2 Dimensional Protein Electrophoresis

1. Definition

   • Proteins are separated based on 2 factors, in 2 dimensions, to allow us to better track why the sample ran differently.

2. Dimension 1: Isoelectric Focusing Gels

   • Requires a gel with non-diffusing pH gradient
     • Ampholytes
       • Molecules that can buffer at their pl
       • A series of ampholytes are embedded into the gel
         • Allows them to maintain a pH gradient without moving during electrophoresis
           • Ampholytes can still move by diffusion, so gradient stable but not permeable.
     • Proteins will be separated by their overall pI: The average of pI of all ionizable groups
       • Amino and Carboxyl ends
         • R groups of charged AA
     • Proteins are electrophoresed through the ampholyte- containing gel until they hit their pI.
       • At the pI they become uncharged and STOP
         • Can take 12-72 hours

3. Dimension 2: SDS- Page

   • Isoelectric focusing strips are treated to denature proteins and coats with SDS
     • Uniformly negative proteins are next resolved by size
   • Can rapidly separate thousands of proteins in a mixture to individual spots
     • Samples from different sources can be compared to determine what proteins are different
       • Species A vs B
       • Tissue X vs Y
       • Normal vs Diseased

4. Band Identification

   • No matter what type of gel is used, a protein blob/band has few visually identifying characteristics
   • To know for certain band you are interested in studying is present/absent/moved/ other you need a way to detect it specifically
   • This may be done using antibody binding reactions or spot protein sequencing