Long story. I can't use any normal endogenous control in for my westerns, so I decided to go with a whole protein stain with amido black. The amido black solution I purchased has isopropanol in it and it seems to react with the membrane (0.15 amido stain, 10% acetic acid 25% isopropanol). The second I put on the stain solution it shrinks my membrane and warps it. I did a test with and without the methanol and the shrinking is due to the methanol in the transfer buffer. I also tried drying the membrane and rinsing several times in milliq. The easiest thing would be to make my own stain made up in methanol, but the other way I image would be using isopropanol to matrix match the stain solution. Thoughts?
Dear Brittany, thank you for sharing this interesting problem with other RG members, and sorry to see that your question has not yet received any expert answers. As an inorganic chemist I'm absolutely no expert in this field and I'm not in the position to provide a qualified answer. However, I made the experience that it often pays off to search the "Questions" and "Publications" section of RG for related questions and useful literature references. For example, please have a look at the answers given to the following closely related technical question which has been posted earlier on RG:
Can we do western blot transfer without methanol in the transfer buffer?
https://www.researchgate.net/post/Can_we_do_western_blot_transfer_without_methanol_in_the_transfer_buffer
(43 answers)
Good luck with your research and best wishes, Frank Edelmann
Hi Brittany Karas . Is there a reason for not using Ponceau S for your blot? If you cannot use Ponceau, you can dilute out the Amido Black 1:5 with water.
Also, PVDF membranes handle solvents better than nitrocellulose.
Frank T. Edelmann thank you for your suggestion. I had seen that question already, but I didn't find the response to be relevant enough for my purpose.
I am unsure, I think you may but only under certain circumstances and one of them (or the most important one ) is the protocol.
Brittany Karas Isn't it cool to have friends on RG that upvote all answers no matter how helpful they are?
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