ECL Western troubles

Curt Ashendel ashendel at aclcb.purdue.edu
Wed Sep 18 16:15:20 EST 1996


On 2 Sep 1996 16:07:46 -0700, Jose Bonner  <jbonner at bio.indiana.edu > 
posted to bionet.molbio.methds-reagnts:

>We're having trouble with Western blots probed by ECL.  Films sometimes 
>come up blank, even though antibodies and reagents are OK.  Sometimes a 
>5-minute exposure is fine, but a 1-hour exposure (started just after) is 
>blank--not even any background.  What can interfere with ECL?  The 
>problem comes and goes; good blots interspersed with blanks.  Reprobe 
>the blanks, and sometimes they come up good; reprobe the good ones, 
>sometimes they come up blank.  Anyone else have this problem?

Unfortunately, the cause of this could be just about anything, 
depending on how careful you (and your lab) are about following 
procedures carefully and how you share reagents, equipment, etc. You 
have to narrow down the possibilities by comparing a failure to a 
success and identifying what has changed and not changed between 
attempts. Combining several comparisons should help reduce the 
possibilities. 

One experience we had in the distant past that gave us similar 
irreproducibility was the our reuse of antibody solutions. When we 
switched to *always*  making a fresh dilution of 
antibody/antiserum/protein-A, etc. nearly all of our problems 
disappeared. Apparently these solutions get depleted or go bad due to 
microbial growth, and they do so unpredictably (sometimes after only 
one use on a blot). 

We also never reuse ECL solutions, and we use a fixed amount of ECL 
solution (1.5 ml per 11x16 cm blot) that is just enough to 
completely wet it, then we discard the excess that drips off.

This always works, even for novices. Any failure is attributable to 
bad gels, blotting, samples, or antibody (all easily controled for).


Curt Ashendel
Purdue University, West Lafayette, IN
ashendel at aclcb.purdue.edu



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