protein passes through membrane!?

Sean Patterson seanpat at FMED2.UNCU.EDU.AR
Mon Sep 20 08:33:29 EST 1999


>Hi all,
>
>I have severe problems with western blotting! I use the standard
>protocol which is used in our lab for YEARS, and suddenly I have
>problems (and I need this blot, for sure, very, very soon!!!).
>The problem is: the proteins doesnt bind to the membrane but passes
>through it, as I can see for the prestained standard, which I can found
>in the bottom layer of whatman paper! I tried to use two layers of
>membranes and shorter blotting times, but is it everytime the same!
>The protocol is: semidry blotting in 10 mM CAPS pH 11.0, 10 % Methanol,
>Nitrocellulose membrane 0,2 µm, 30 min with 90 mA for a minigel.
>
>Who has an idea what could be the reason for this?
>
>Any comments are welcome,
>
>Andreas

Hi Andreas - I've resolved problems with very hydrophilic proteins not
sticking to nitrocellulose by changing to a charge-modified membrane (such
as Immobilon-N, no relation, etc.), but if your standards are going through
it sounds as if the blotting itself is the problem. For blotting out of a
12% x 1.5mm minigel in a semi-dry apparatus, we would use no more than
1mA/cm2 for 30-45 minutes, at pH9 with 20% methanol. Increasing the current
or time would result in the smaller prestained MW standards (which don't
stick as well as regular standards) transferring through the nitrocellulose.

Are you sure that your sample protein isn't sticking? Have you stained the
membrane after transfer with Ponceau S?

Sean


Sean Patterson, Ph.D.
Catedra de Fisiologia, CC33
Facultad de Ciencias Medicas
Universidad Nacional de Cuyo
Mendoza, Argentina
Tel: (0261) 430-9385
Fax: (0261) 449-4117
e-mail: seanpat at fmed2.uncu.edu.ar





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