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
> 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
4170 Graves Hall
333 w. 10th ave
Col. OH. 43235

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