NorthWestern protocol

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Fri Dec 6 11:24:20 EST 1996


In article <Pine.A32.3.94.961205063239.53886A-100000 at umabnet.ab.umd.edu>,
"Mary P. Remington" <mremingt at umabnet.ab.umd.edu> wrote:

> A friend is looking for a protocol for a Northwestern blot.  Any
> references appreciated.  Thanks, Mary


Run your protein gel as usual, blot it to a PVDF membrane, and block for
1h with 1%BSA in 10mM Tris, 50mM NaCl, 2mM EDTA. Try to get good BSA that
does not have excessive RNase activity (RIA-grade is usually fine). Wash
your blot in buffer without BSA, three times for five minutes, then
incubate with RNA (1mio cpm at least) for 30min at RT. You can also try
4C. Then, wash five times with buffer without RNA, dry filter and expose
to X-ray film. 

If your protein has to be renatured to bind, try denaturing in 6M GuHCl in
binding buffer (membrane will become transparent), and renaturing in
steps. Remove half of the buffer every 10min, and replace with binding
buffer without GuHCl. After 10 cycles, remove buffer completely and wash
for additional 10min before blocking.

Hope it helps,
Frank

-- 
Dr. Frank O. Fackelmayer
Division of Biology
University of Konstanz
D-78434 Konstanz
Germany



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