background on 2D western blot

[Araluen Freeman]

Hi Will!

All I can offer is that our system with Western blotting on NC works
consistently.  I use 5% skim milk in TBST buffer (10 mM Tris-HCl, pH 8.0;
0.15 M NaCl; 0.1% Tween-20) to block, and also use the blocking buffer to
dilute the primary and secondary antibody, and for the washing steps
between.

I use a chemiluminescence detection system, and found at first an unspecific
'streak' on my blot.  This turned out to be me applying the strepavidin-AP
directly over my blot (after incubation with the biotinylated secondary
antibody) instead of to the incubation solution, thus removing my blocking
and getting this signature streak *grin*.

Hope you iron out your problem soon.   ;-)

 Araluen


"Will Chiu" <will.chiu at auckland.ac.nz> wrote in message
news:20010928103151.16882.qmail at ww02.jatek.com...
> Dear All
>
> I have been experiencing intense spotty backgrounds on my 2D western blot
> probed with sera using NC membrane. A switch to PVDF membrane managed to
rid
> of the spots but background noise was too great despite of trial of
various
> blocking agents.
> would nylon membrane help?
>
> Any suggestion is welcome.
>
> Help
>
>
>
>
> <http://www.biowww.net/forum/read.php?f=3&i=600&t=600>
>