Which method uses ampholytes for creating a pH gradient?
Isoelectric focusing requires a stable, continuous pH gradient along electrophoretic path for proteins to focus precisely at their pI. Carrier ampholytes are complex mixtures of low-molecular-weight polycarboxylic polyamino amphoteric compounds possessing varied pI values between 3 and 10. Under electric field, they migrate and buffer locally to establish a smooth pH gradient from anode to cathode. SDS-PAGE, capillary zone electrophoresis and second dimension of 2D gels use uniform buffer pH, not gradient. Ampholytes or immobilines are thus central to creating the field for charge-based 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.