ECL problems

Graham Atherton grggta at picr.cr.man.ac.uk
Tue Mar 28 06:17:05 EST 1995


We are encountering a few peculiarities of the Amersham ECL detection
system for Western blots. Any comments would be appreciated:-
1) No bands of any sort  detected, thus we changed from using skimmed
milk to BSA (the skimmed milk was a new batch).
2) Now we got a few faint, specific bands of the 'right' size. Re-
exposure resulted in no bands! Presumably the signal had decayed but
the second exposure was taken only a minute or so after the first!
3)Fresh ECL substrate was applied and this gave two different
results  i)Many more specific bands appeared - including those
that had appeared the first time - and these bands were much stronger,
giving a good result. Perhaps we should always apply substrate twice?
        ii)However, one of three blots probed simultaneously now had
high background, giving a completely black exposure. Specific bands
were apparent on this blot, just, when holding it up to the light.

  So although reapplying substrate gave us a good result 2/3 times,
it also gives an unusable result 1/3 times. This makes this system
unattractive to us for further use - it is too unreliable.
  Can any ECL stalwort suggest how we can improve the reproducability
of our experiments?
Many thanks
Graham Atherton
grggta at picr.cr.man.ac.uk



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