What separates molecules in the first dimension of 2D electrophoresis?
Two-dimensional electrophoresis couples orthogonal separation principles to resolve highly complex proteomes containing thousands of polypeptides. First dimension is isoelectric focusing in immobilized pH gradient strips or carrier ampholyte tubes where proteins migrate under electric field until net charge becomes zero at their isoelectric point, effectively focusing at pI independent of size. This separates isoforms differing by charged modifications. Second dimension is SDS-PAGE where focused proteins are further separated by molecular mass. Charge alone or solubility does not define focusi
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.