Hi guys, I'm confused and will really appreciate your comments. Here's my story:
We've run out of methanol in the lab and have been waiting on the new order to be delivered for longer than a week now. As we did not want to stop experiments in the meantime, we decided to use ethanol instead for the transfer buffer (Tris-Gly + 20% Ethanol) and proceed as normal.
Now, here comes the problem. Transfers seem to be working fine and canít really see a difference with the ones we did with 20% methanol. We've had successful blots and equivalent results when using an anti-lysozyme antibody (this is a polyclonal made in rabbit). BUT, we are failing to detect beta-actin with a monoclonal made in mouse. We have successfully used this antibody before, and as I said the ONLY change is been the ethanol for methanol in the transfer buffer. I must point here that the samples for lysozyme and actin detection are different but again, we have previously tested both successfully.
With these results my conclusion is that the ethanol is somehow having a detrimental effect in the immunodetecion of Actin but not Lysozyme. Is it possible that ethanol is "selectively" altering some proteins but not others? Is it the antibodies? As the anti-actin is a monoclonal, do you think maybe the ethanol is affecting the epitope that is being recognised in the actin protein, and even if some changes happen in Lysozyme the polyclonal antibodies will still bind to unaltered epitopes?
We are hoping to get methanol delivered today, and we'll get back to our usual protocol, but I'm curious as to why this is happening. Your thoughts will be greatly appreciated!!
ethanol instead of methanol in transfer buffer
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