Chemiluminescent detection

Ted M. tedm at darkwing.uoregon.edu
Mon Sep 18 23:47:46 EST 1995


In article <9509151702.AA19579 at bnlux1.bnl.gov.bnl.gov>,
berges at BNLUX1.BNL.GOV (John A. Berges) wrote:

> Hi Folks,
...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
>  
John,
   Normally the ECL system works wonderfully, I have a similar protocol
with Triton and SDS in the washes and it is very nice; low background and
sensitive.
The secondary antiboby is in a high BSA solution and there is a tendency
to add a little azide to some of these solutions, could this be your
problem? Azide apparently chelates to the Fe in the heme of HRP and
doesn't like to be washed out, giving strong inhibition. Check if someone
added some; I've been in protein purification labs where they put  azide
in everything except the coffee!
Good Luck!
 regards,

Ted Michelini
Institute of Molecular Biology
University of Oregon
tedm at darkwing.uoregon.edu



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