Chemiluminescent detection

John A. Berges berges at BNLUX1.BNL.GOV
Fri Sep 15 12:04:22 EST 1995


Hi Folks,

I have a western-blotting problem for which I hope someone out there has a
solution, or at least some advice.

I have an polyclonal antibody raised in rabbit to nitrate reductase from a
marine microalgae.  Using a goat-antirabbit-alkaline phosphatase secondary
antibody, I can detect the protein on a nitrocellulose blot without any
trouble (BCIP/NBT colour reaction), through for low protein quantities
scanning the blots gives me rather high variability.   

Since the chemiluminescent detection using Amersham's ECL kit and a
horseradish peroxidase secondary antibody has been working very successfully
(very clean backgrounds and wide linear range of detection) for our lab
using another primary antibody, I tried it and got lovely results...ONCE!

Since then I have had no luck at all.  Often I get nothing (no background
either), other times I get odd patterns on the blot; half the lanes show up,
or I get bare patches in the middle. It seems unlikely that this is a
primary antibody problem...after the ECL detection fails, I can still add AP
antibody and get good results on the same blot.  Therefore the problem
appears to lie with the HRP secondary antibody.  I have several checks:  the
HRP stocks are in good shape (they work on a related antibody), and the
detection reagent are OK also.  One concern I do have is our washing buffer:
it contains Tritox X-100 and a small amount of SDS (I know they say you
aren't supposed to use these, but we have always had very nice results, i.e.
lower backgrounds than using Tween) and I have some concerns that it might
be inhibiting the HRP reaction somehow (we wash extensively in TBS after the
washes).  But then why did it work once??

I'd appreciate any comments.

Thanks.

          
________________________________________________________________
      _/_/_/     _/      _/   _/    Dr. John A. Berges
     _/    _/   _/      _/   _/     Oceanic and Atmospheric
    _/    _/   _/_/    _/   _/      Sciences Divison
   _/_/_/     _/ _/   _/   _/       Brookhaven National Lab
  _/     _/  _/   _/ _/   _/        Upton, NY 11973        
 _/     _/  _/    _/_/   _/         Phone: 516-282-4077
_/_/_/_/   _/      _/   _/_/_/_/    Email: berges at BNLUX1.BNL.GOV
________________________________________________________________




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