ECL detection: more comments...

echeverri at sci.wfeb.edu echeverri at sci.wfeb.edu
Wed Jul 28 10:57:16 EST 1993


In article <1993Jul27.152215.5488 at crc.ac.uk>, dmartin at crc.ac.uk (David Martin x3175) writes:
> 
> We have had excellent results using Amershams ECL with a variety of western
> blot types (Ab-HRP, Ab-Biotin:avidin-HRP, ligand-biotin:avidin-hrp)
> 
> Just wash in TBS or similar (ie no gelatin) prior to adding the ECL reagents
> after your usual probing method and you should get good results.
> 
> best of luck
> 
> ....d

I would like to second this: I've been using Amersham's ECL detection 
system for years, and it is great.  However, I'd like to share a disturbing 
observation which has cost me much time and effort, in the hopes of sparing 
you netters some ulcers.  I was using a biotinylated secondary, in 
conjunction with Amersham's Biotinylated-Streptavidin-HRP complex to boost 
sensitivity on blots of rat tissue high-speed cytosols.  Most tissues were 
showing beautiful bands in the 75kDa range (NOT expected with my primaries)
which turned out to be due to the Biot-Strept.-HRP complex (thus the 
importance of running parallel secondary (and tertiary in this case) control 
blots! :-*).  I subsequently came across a possible explanation for this: 
it seems that streptavidin contains the sequence RYD which is thought to 
mimick the RGD recognition sequence for adhesion receptors.  Has anyone 
else had this problem?  If so, I'd be interested in knowing the types of 
tissue/extracts which proved troublesome, to avoid this in the future...
(may post a summary depending on interest generated)

Chris E.
Worcester Foundation
  for Experimental Biology  
Worcester, MA



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