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not completed transfer proteins on NT membrane - western blot (Oct/19/2010 )

I changed nitrocellulose membrane pore size 0.45 um to 0.2 um. (also changed the company)
After I transfered, I found proteins some times completed transfering
but most of experiments the protein often detached. (see pic.)
I don't know why ?? what is the critical process ?
How should I do ?

Thank you
Attached Image


you may not be completely filling the nitrocellulose pores with (water, buffer). before use, we float the dry membrane on top of water and let it sit until water seeps through and coats the top.

(to oversimplify) keep in mind that the larger the protein, the less complete the transfer.


Thank you mdfenko
This time, I extended the time for soaking the membrane (and also changed from 20%MeOH in water , the recommended from data sheet, to Transferring buffer only ). The result was nice

Thank you very much