ECL Western Blotting, need help

Alix Helme-Guizon ahelme at lovelace.infobiogen.fr
Mon Aug 25 03:30:47 EST 1997


Hi,
i use routinely PVDF (Immobilon) membrane without troubles with background
since i have increased the time of washing after the second antibody; this
appears to be a crucial point for removing the back ground. i use the
secondary antibody at 1/10 000 RT, 3 hours and then at least five washing
of 30 min each ( an all afternoon only for the washings). then an ECL
detection is performed. avoid supersignal if you have problmes with the
background, because it is higher with this method than with a classical
ECL
 
 Don´t wash for
> extended times, better change buffers more often (I discard the buffer of
> the first wash usually after 1-2 mins.)
do both!

good luck
alix




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