ECL and Western Blots

Kevin Morano ez005528 at ucdavis.edu
Sun Aug 1 14:22:24 EST 1993


Helmut Dotzlaw (dotzlaw at ccu.umanitoba.ca) wrote:
: In article <17906 at news.duke.edu>, lees at acpub.duke.edu (Steve Lee) wrote:
: > 
: > I was wondering if anyone out there has used Amersham's ECL kit for
: > western blot detection... is it as sensitive as they claim it is?
: > 
: > 
: I have.  I'm not sure if it's as sensitive as they "claim", but I can say
: that it is definitely much more sensitive than AP.  From zero signal on AP
: to a "blasting" exposure using the ECL.  It's less work than AP, too.  We
: use it all the time now.

: > also, what's the maximun amount of protein that someone has successfully
: > loaded onto Bio-Rad's Mini-Protean II?
: >
: I've gone up to 500 ug on a 1.5mm gel using the 10-well spacers. 
: > 
: Cheers,

: Helmut

Our lab has used both Amersham's ECL and NEN's Rennaisance kits.( Our reps
tell us they are they EXACT same things) We get excellent results from
both, usually using the primary polyclonal antibodies at 1:20K to 1:50K
and the secondary at 1:30K. You'll notice the huge dilutions--not only
possible but essential. If you do a 1:2000 with a standard primary, you'll
be able to read by the light given off from each lane. The detection level
is quite low as well, lighting up rare proteins from approx. .1 OD of
cells. 
Also,I have loaded about 200 ug on the BRMPII using the 1.5 mm prep comb,
no problem. Good Luck.
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Internet:kamorano at ucdavis.edu     "Why does it happen, because it happens..."
Bitnet:kamorano at ucdavis                                              N. Peart
Section of Microbiology, University of California, Davis



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