the protein is a transcription factor subunit of AP1, the sample was treated with B-ME and boiled at 95 degrees for 5 min, ran on SDS-PAGE, could it still be in a dimer after all this?
It is possible that some sort of a cross linking is happening hence the higher Mw. Try 2-3 min of heating instead of 5 min? Also reduce B-ME concentration. What BME concentration are you using?
There are some proteins that retain oligomeric state through SDS-PAGE, but another possibility is that you are detecting another protein due to insufficient specificity of your antibody.
I use 5% BME in the loading buffer and an anti FRA1 monoclonal antibody from Cell Signalling.. and the band i get with the higher MW. Is only in one of my treated conditions and not on the control or any other conditions that i ran in the same western blot so i think its specific..
I also agree with Adam. If you have epitope that can cross react that could form false positive. However you are using monoclonal Ab which means cross reactivity can be ruled out?. In any event Try to reduce the primary Ab concentration?
Oligomeric state not due to sulfur bonds are usually lost with SDS and heat.
Therefore my prediction is: you are forming nonspecific intra S-S bonds.
More importantly 5% BME might be too much. Try to use 1% BME or DTT. Good luck.
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