Separation of porteins is based on the migration of charged protein in an electric field
The migration of a protein in a gel during electrophoresis is a function of its size and shape
m = V/E = Z/ f
M: electrophoretic mobility
V: velocity; E: electrical potential
Z: net charge; f: frictional coefficient
SDS-PAGE: Sodium Dodecyl Sulfate (SDS) Polyacrylamide Gel Electrophoresis
SDS binds to most proteins probably by hydrophobic interaction. One SDS for every two AAs, Thus, each protein has a similar charge-to-mass ratio.
Coomassie blue stains protein. Western blot
Estimating the Molecular Weight of a Protein
Isoelectric Focusing
pI of a protein: net charge=0
A pH gradient is established by allowing a mixture of organic acids and bases (ampholytes). Protein migrates until it reaches the pH that matches its pI
Two-Dimensional Electrophoresis
Separates proteins of identical MW that differ in pI or proteins with similar pI but different MW.
Activity Vs. Specific Activity
