Best Technique to Study Protein Half-Life in Animal Cells

Which of the following techniques should be used to study the half-life of protein A in an animal cell that can be after labelling all proteins being translated with a radioactive amino acid for a short duration followed by removal of radiolabel and growth of cell in normal medium.

A. SDS PAGE followed by autoradiography
B. SDS-PAGE followed by Western blotting
C. Immunoprecipitation followed by autoradiography
D. Immunoprecipitation followed by SDS-PAGE and autoradiography

Best Technique to Study Protein Half-Life in Animal Cells

Correct Answer: (D) Immunoprecipitation Followed by SDS-PAGE and Autoradiography

Studying the half-life of a protein requires a technique that can specifically track the decay of a labeled protein over time. In this experiment, all proteins are initially labeled using a radioactive amino acid pulse (pulse-chase experiment), followed by growing the cells in a non-radioactive medium (chase). The best method to track the degradation of Protein A is Immunoprecipitation (IP) followed by SDS-PAGE and Autoradiography.

Why is Option (D) the Best Choice to  Study Protein Half-Life in Animal Cells ?

1. Immunoprecipitation Ensures Protein Specificity

   • Since all proteins are initially labeled, a specific purification method is required to isolate only Protein A.
   • Immunoprecipitation (IP) achieves this by using antibodies specific to Protein A, allowing selective extraction from the complex cellular mixture.

2. SDS-PAGE Helps Analyze Protein Size and Degradation

   • After immunoprecipitation, SDS-PAGE is performed to separate the protein based on its molecular weight, enabling comparison across different time points.

3. Autoradiography Detects the Labeled Protein

   • Since Protein A was originally labeled with a radioactive amino acid, autoradiography can track its presence and decay over time.
   • A gradual decrease in the autoradiographic signal indicates protein degradation and half-life estimation.

Why Other Options Are Less Suitable?

• (A) SDS-PAGE followed by Autoradiography:

  • This method detects all labeled proteins, not just Protein A, making it non-specific.

• (B) SDS-PAGE followed by Western Blotting:

  • Western blot detects total protein levels, but it cannot distinguish between newly synthesized vs. pre-existing proteins in pulse-chase experiments.

• (C) Immunoprecipitation followed by Autoradiography:

  • While this isolates Protein A, without SDS-PAGE, we cannot confirm if degradation products exist.

Conclusion

To measure Protein A’s half-life, Immunoprecipitation followed by SDS-PAGE and Autoradiography is the most precise technique, ensuring specificity, molecular weight confirmation, and detection of radioactive decay over time.