Separation and characterization of proteins by gel electrophoresis

Principles:
All proteins have a characteristic isoelectric point (pI) and a molecular mass (mol wt = grams/mol). Both parameters are dictated by the amino acid composition of that protein.

Physico-chemical basis for protein separation:

When a mixture of proteins is placed in an electrical field (i.e., subjected to a current) at a fixed pH, each protein will migrate either toward the anode (+) or the cathode(-) depending on the proteins net charge.

The rate of migration of each protein is determined by the degree of physical resistance it encounters as it moves though the gel medium (e.g., hydrated polyacrylamide gel matrix).

Therefore, small proteins will migrate faster than larger proteins, and the distance of migration though the gel matrix will be inversely proportional to the size of the protein.

SDS-PAGE: Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis All proteins acquire a net negative charge by binding SDS. All proteins will migrate toward the anode (+) at a rate based on their mass.