Westerns

David Ward Rusnak rusnak~dw at glaxo.com
Fri Feb 9 07:58:56 EST 1996


In article <v01510101ad367da8470f@[129.112.20.59]>, clark02 at UTSW.SWMED.EDU
(Kathleen Clark) wrote:

>  Dear cell biologists,
> 
> I'm not sure if this is the most appropraite place to post this inquiry.
> I,ve tried to compare Western results using the nitrocellulose/hrp based
> ECL kit and the PVDF/alk-phos based Tropix kit. The ECL kit produces a
> large number of nonspecific bands that I can't eliminate using conventional
> measures (long blocking, dilute antisera and lowering amount of sample on
> gel). The Tropix kit does not show these nonspecific bands but it takes so
> long to get a strong signal that the overall background it fairly high. Has
> anyone reached a happy medium concerning these two products ? Also, I
> should note that the protein source for these experiments is Drosophila
> ovaries.
> 
> 
> Thnaks in advance,
> 
> Kathleen Clark
> clark02 at utsw.swmed.edu

KC,

I tend to agree with the other replies.  The secondary may well be the
culprit.  
In the HRP ECL kit, what secondary Ab are you using and who is the
manufacturer? We have been using Amershams ECL kit for years.  The ECL
reagents are fine, but quite frankly, we find their secondary antibodies
(the ones that come with the kit)  are average at best.  We started using
secondaries from Jackson Immunoresearch in West Grove PA  (I have
absolutely no financial stake with them whatsoever, they simply have the
best secondaries in the business, IMHO) and the ECL's were much cleaner
and "hotter."  

For doing antimouse IgG detection, we use an HRP conjugated donkey
anti-mouse antibody at a dilution of 1:10,000

My $0.02,

David



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