I'm carrying out B-cell isolation from mouse tissue by cell sorting using 2x biotinylated probes. One is a wild-type protein and the other has a single point mutation to reduce binding of B-cells to a specific epitope, so by using these 2 probes together I can find B-cells which target only the specific epitope I'm interested in for analysis.
The wild-type probe was working fine, but I wasn't seeing any binding of cells to the mutant protein in the test sort I've done so I ran a quick SDS-PAGE to check it. It looks like the protein just completely disappeared (fell apart, degraded) under reducing conditions (gel attached) and I have no plausible explanations for why this would occur in the mutant but not the wild-type protein, given that it's only one amino acid different from the wild-type. The non-reduced lane looks to be identical in size to my wild-type reference.
I have sequenced the plasmid used for the transfection and the read (while it doesn't cover the entire sequence) comes back clean and 100% identical to the reference. ELISAs I've done with it using tool antibodies bind as expected (although slightly lower level than the reference). If it was just a bundle of non-specific protein I'm thinking that I would see binding from my negative control and also the antibody which targets the knocked-out epitope.
Any answers or advice would be much appreciated as doesn't make any sense to me.
Its not at all unreasonable for a single point mutation to have a profound impact on a proteins conformation and stability, see the well characterized E->V mutation in sickle cell anemia. Also there are "conservative" mutations (I->L) and more extreme ones that could be predicted to have a greater effect on structure, like a proline in the middle of a predicted a-helix, which is yours? Also it looks like a high MW smear is present in the mutant reduced lane, is this a consistent finding? Why was this AA chosen to mutate?
Thanks for your answer. To answer your queries:
Since the ELISA confirms that this protein is your mutant and not a non specific protein, the only other explanation for a missing/degrading protein on the gel is protease contamination. Was the same reducing buffer used for all three reactions ? Consider performing a run where you have supplemented the buffer with protease inhibitor cocktail during incubation.
Have you repeated this experiment with a batch of freshly purified mutant protein ?
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