Western problems

Tobias Straub tobias.straub at mail.uni-wuerzburg.de
Wed Nov 18 08:41:51 EST 1998


In article <364C993A.43E5E7D1 at york.uchsc.edu> , kurzbw at YORK.UCHSC.EDU 
wrote:

>1.  I am using PVDF membranes for Westerns.  After ECL, I have very high
>background with "negative" bands where the proteins were visualized with
>previous 20% methanol staining.

the methanol staining should not be the problem if the membrane is
completely methanol-activated before immuno detection. therefore I believe
your background is most prabably due to insufficient blocking (try another
reagent like e.g. casein) or too high concentration of secondary antibody.

>2.  How many times can PVDF membranes be stripped?  And after stripping,
>can proteins still be visualized with 20% methanol staining?

it depends on the amount and kind of protein that you want to detect.
stripping can be done for more than 10 times if you have a lot of protein
and if this protein sticks tightly to the membrane.
I don't think that you still can do the methanol staining after stripping,
because there will be a lot of blocking protein left on the membrane.
one question: why do you do the methanol staining? in my opinion this is not
a very worthy method. if you just want to check if some protein transferred
you can look at the membrane immediately after taking it out of the
aparatus. if you look at a certain angle you will some bands. and if you
have a lot of protein you will see the bands as the membrane dries. whenever
I want to know if the transfer was ok, I run an extra lane and stain it with
amido black or indian ink. 

>3.  What are the real life pros and cons of PVDF vs. nitrocellulose?

after having done 100000 blots I believe that there are no pros of NC. PVDF
is more reliable, easier to handle, easier to strip, has lower background.
OK, one contra: its more expensive.


tobias


------------------------------------------------
Tobias Straub
Medizinische Poliklinik, University of Wuerzburg
Klinikstrasse 6-8, 97072 Wuerzburg, Germany
Phone: +49-931-2017085      Fax: +49-931-2017120
Tobias.Straub at mail.uni-wuerzburg.de
------------------------------------------------



More information about the Methods mailing list