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#pI value

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Electrophoresis

In isoelectric focusing, separation is based on:

Isoelectric focusing separates macromolecules based on differences in isoelectric point rather than solely molecular weight or total charge at fixed pH. A stable pH gradient is created using carrier ampholytes or immobilines immobilized in gel matrix. Proteins migrate under electric field until reaching zone where local pH equals pI, resulting in zero net charge and cessation of movement. This focusing concentrates proteins into extremely narrow bands at characteristic pH values. Distinction from SDS-PAGE which separates by size, or ion-exchange which separates by constant charge, underlies it

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.

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Electrophoresis

In IEF, where will a protein stop migrating?

In isoelectric focusing, a stable pH gradient is established using carrier ampholytes or immobilized buffers spanning acidic to basic range. Proteins introduced into gradient acquire positive charge below their pI and negative charge above, causing migration in electric field. As movement proceeds, net charge diminishes upon approaching pH equal to isoelectric point, where protein becomes electrically neutral. Loss of electrophoretic driving force halts further migration, while diffusion is counteracted by focusing effect, concentrating proteins into sharp zones at characteristic positions. Fo

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.

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