I've run plenty of westerns and never had this happen. We do a Towbin buffer at 4degrees o/n and PDV membrane. Using exact steps I've used before, set it up and it was still running when i stopped it, so it wasn't a power failure. When I opened there was a reddish-brown scum in the buffer, which immediately suggested something amiss. I double checked orientation--fine. I stained gels and membranes. Aside from a very small amount of high molecular weight proteins, the gels were empty, but so were the membranes. The only change from the Western I did at the beginning of the week (aside from samples) was a new bottle of Towbin buffer made by a student. What ingredient might have been left out or maybe too much added?
Could it be something else?
Hi,
I think you need to check the following to know the cause of the problem.
1.The voltage applied and time of transfer(if the voltage is too high, it might penetrate through the membrane)
2.Amount of methanol in your transfer buffer(excessive methanol could be a cause).
3.The foam pad....if changing colour could cause the scum .
4.Air bubbles during the transfer from gel to membrane.
5.Does the membrane and gel make a proper contact during blotting.
I hope this could provide an idea of what the cause might be.
All the best!
Thanks. I'm pretty sure it was the buffer. A student made it and on questioning didn't add things in best order. All other components had been used by me literally 2days before.
I think you already answered your question by yourself. Never use buffers prepared by students without your supervision for important experiments.
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