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No transfer of protein on PVDF membrane - Western blot troubleshooting (Oct/11/2005 )

I started western blot for detection a 50 KDa protein. At first the transfer was perfect and after staining with ponceu I could see nice bands on the membrane. But now I can not see anything after staining the membrane. When I used Coomasie blue for staining the gel all the proteins were there. All the conditions are like before and I did not change anything. I use 1X transfer buffer for 1 hr with 100 voltage.
I am really surprised what happend?
I would be very glad to receive your recommendations and opininons for my problem.


you can aldehyde-fix your proteins to the membrane right after transfer, if you use PVDF?

or are you losing your stuff during the transfer?


Thats a strange problem... but all I can say is that somehow your conditions are not the same (specially when you say that if you stain the gel everything is still in there)... How many times has this happend to you?

Check that current is really leaving the power supply... is not enough for the voltage to up, the current also needs to up...


If you failed to put methanol in the transfer buffer, this might happen. The other circumstances under which I can see this happening are if there was no current running -- either due to a faulty power supply, incorrect assembly of the transfer apparatus, or the transfer apparatus is broken (one of the wires is snapped)...


pH your buffer?

maybe there was a mistake in the latest batch of transfer buffer?


Do you re-use the transfer buffer? It happened two me once then I reallized I had used transfer buffer for so many times..... After making new buffer everything went back to normal.

A pre-stained marker could help you to monitor transfer status easily