background on 2D western blot

Araluen Freeman araluen_f at
Tue Oct 2 11:41:58 EST 2001

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

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> wrote in message
news:20010928103151.16882.qmail at
> 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
> of the spots but background noise was too great despite of trial of
> blocking agents.
> would nylon membrane help?
> Any suggestion is welcome.
> Help
> <>

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