Which of the following approaches is used to measure the approximate mass of the purified protein complex and
to determine its subunit composition?
(a) Polyacrylamide gel electrophoresis and molecular cloning
(b) Ion-exchange chromatography and density-gradient centrifugation
(c)Genomics and recombinant DNA technology
(d)Gel-filtration chromatography and Western blotting

The correct answer is:

(d) Gel-filtration chromatography and Western blotting

Explanation:

1. Gel-filtration chromatography (Size-exclusion chromatography, SEC)

   • Separates proteins based on size and approximate molecular weight.
   • Larger protein complexes elute first, while smaller ones elute later due to differential movement through porous beads.
   • Helps estimate the mass of a purified protein complex.

2. Western blotting

   • Detects specific proteins using antibodies.
   • Can help determine the subunit composition of a protein complex if individual subunits are resolved on a SDS-PAGE gel and probed with specific antibodies.

Why Not the Other Options?

• (a) Polyacrylamide gel electrophoresis and molecular cloning →

  • PAGE (SDS-PAGE or native PAGE) helps analyze protein size but is not the best method for determining the approximate mass of an intact protein complex.
  • Molecular cloning is for gene manipulation, not protein mass analysis.

• (b) Ion-exchange chromatography and density-gradient centrifugation →

  • Ion-exchange chromatography separates proteins based on charge, not mass.
  • Density-gradient centrifugation is more useful for nucleic acids or organelles rather than purified protein complexes.

• (c) Genomics and recombinant DNA technology →

  • These are used for studying genes and producing recombinant proteins but do not determine protein mass or subunit composition.

Conclusion:

Gel-filtration chromatography provides an estimate of protein complex mass, while Western blotting helps analyze subunit composition, making (d) the correct answer.