Q.8 Two monomeric His–tagged proteins of identical molecular weight are present in a solution, pls of these two proteins are 5.6 and
6.8. Which one of the following techniques can be used to separate them?
(A) Denaturing polyacrylamide gel Electrophoresis
(B) Size–exclusion chromatography
(C) lon–exchange chromatography
(D) Nickel affinity chromatography
Ion-exchange chromatography effectively separates these two monomeric His-tagged proteins due to their differing pIs of 5.6 and 6.8. Proteins have identical molecular weights, so techniques relying on size cannot distinguish them, but their pI differences allow charge-based separation.
Option Analysis
Denaturing PAGE (A) separates proteins solely by molecular weight after SDS denaturation imparts uniform negative charge proportional to length. Since both proteins have identical molecular weights and His-tags add negligible mass, they co-migrate indistinguishably.
Size-exclusion chromatography (B) resolves proteins by hydrodynamic volume/size in native or denatured states. Identical molecular weights mean similar sizes, preventing separation regardless of His-tags or pI.
Ion-exchange chromatography (C) exploits net charge differences at specific pH. At pH 6.2 (midpoint between pIs), the pI 5.6 protein is more negatively charged (binds anion exchanger stronger), while pI 6.8 protein is less negative or neutral, eluting differently with salt gradients. Correct choice.
Nickel affinity chromatography (D) binds both His-tagged proteins equally to Ni-NTA via polyhistidine, eluting together with imidazole. No discrimination based on pI or size.
This GATE Biotechnology 2020 question tests protein purification principles central to IIT JAM, CSIR NET, and biotech research. His-tags enable affinity purification but not isoelectric separation; pI differences demand ion-exchange for downstream applications like enzyme kinetics or structural studies.
