western blot

Hany Saqr helsayed at magnus.acs.ohio-state.edu
Mon Mar 17 19:10:07 EST 1997


In article <33287A1C.7F1 at BIOLOGY.Watstar.UWaterloo.CA>, M Allen
<M3ALLEN at BIOLOGY.Watstar.UWaterloo.CA> wrote:

> Keld Sorensen wrote:
> > 
> > Depends on the detection system. If you use HRP both are equal, if you
> > use AP, you should use TBS since phosphate is an inhibitor of
> > PHOSPHATASE.
> 
> Hello netters
> I'm relatively new to Western blotting, but our lab routinely uses PBS
> and an AP detection.
> The PBS is soaked out first however.
> Would it make a significant difference to switch to a TBS system?
> Thanks in advance
> Mike Allen
We get good western blots by blocking with 5% milk in TTBS (0.05% tween in TBS) for 1 hour.  Incubate with 1ry Ab, at 1:2000 dilution,at -4C for 16 hours (O/N). Wash with TTBS for several hours, at least five times, 20 minutes each. Incubate with secondary 1b, 1:10000 dil, at room temperature and then detect it with your system (we use ECL).
-- 
Hany Saqr M.D., Ph.D.
Pathology Depat
OSU
4170 Graves Hall
333 w. 10th ave
Col. OH. 43235



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