Electrophoresis done under pH gradient is
High voltage Electrophoresis ( Isoelectric) →Protein move under the influence of applied current as primary gradient in the presence of secondary gradient pH (= Isoelectric focusing)
1. Well suited for separating the components of complex mixtures of protein
2. IEF (isoelectric focusing) is used in conjunction with SDS-PAGE, which separate polypeptides based on PI (isoelectric point) in one dimension and based on Mr in the second.
a. Isoelectric focusing (IEF) → Ionic buffers called ampholytes and an applied electric field are used to generate a pH gradient with a polyacrylic amide mixture. Applied proteins migrate until they reach the region of the matrix where the pH matches their isoelectric point (p1), the pH at which a peptide’s net charge is zero.
b. SDS-PAGE (=Sodium dodecyl sulphate — polyacrylamide gel electrophoresis)
c. Most widely used method for determining the purity of protein (recombinant protein)
d. Gel was stained with Coomassie blue.