Question

In an experiment you perform the following- you attach a flag epitope to HDAC, you over express a mutated Mad1-pro gene within the experimental cell, you immunoprecipitate the protein complex using flag epitope and run that on a non-denaturing SDS PAGE and western blot. Now you add antibodies for HDAC, MAD1 and SIN3A stripping after each addition. What will you observe?

(a) Bands at same position every time

(b) 3 bands at different positions one at a time

(c) Bands at same position on 2 occasions

(d) No band

I have been asked this question during an interview.

I need to ask this question from Transcription in Eukaryotes : Chromatin Remodelling topic in section Gene Expression : Transcription Prokaryotes of Cytogenetics

Answer 1

The correct answer is (c) Bands at same position on 2 occasions

Easy explanation: As Mad1-pro can’t bind to SIN3A due to the mutation it will not precipitate with the HDAC-SIN3A complex. Due to non-denaturing nature of the gel both the proteins migrate together to the same distance so the band appears at the same position.