ECL problem

h8719263 at hkucc.hku.hk h8719263 at hkucc.hku.hk
Tue Apr 18 03:12:47 EST 1995


I have been using Amshersham's ECL as a substrate for my Western. I'm happy
with its sensitivity, but I have had serious problem with it recently. Hope
you can help. My expt is like this. SDS-PAGA under reducing condition, 
electroblot to nitrocellulose (sigma) filters (overnight at 260V using Towbin's
buffer), blocking in TBS with 5% milk and 0.5% tween20 for about 2 hours, 
incubation with primary antibody (ascites diluted in blocking buffer) overnight 
at 4 degree C, wash with blocking buffer, secondary antibody (Dakopatt
rabbit anti-mouse Ig-peroxidase, diluted in blocking buffer), wash, and ECL
reaction as described in the product booklet.
The problem is: in 9 out of 10 expts my blots are very very dirty. And the 
'dirt' is in the form of well-defined lines or 'scratches' across the filters.
The line pattern is usually continuous, even I cut the filters into different 
strips for different antibodies. The problem doesn't seem to be due to
inadequate blocking and washing (as we did that excessively but it happened),
nor is it due to physical damage to the nitrocellulose itself (as we have
handled the filters as gently as possible). 
I have already run out of ideas. If you have any suggestions please email me
or post here. Thanks in advance.

Lam Yun Wah




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