Problems with Westerns

Helmut Dotzlaw dotzlaw at ccu.umanitoba.ca
Thu Jul 29 15:51:20 EST 1993


In article <238v67$98k at samba.oit.unc.edu>, plc at med.unc.edu (Philip L. Carl)
wrote:

>      Recently we have switched to enhanced chemoluminescense
> to detect proteins on Western blots and have been generally
> pleased with the increased sensitivity.  However our blots
> often appear "blotchy" with irregularly shaped dark regions.
> We believe this is due to small amounts of gel that adhere
> tenaciously to the blots after semi-dry transfer, although
> we haven't ruled out that there may be other causes.  I was
> thinking of putting a little glycerol into the transfer
> buffers to make the membrane a little slippery.  The problem
> seems to be worse with nitrocellulose compared to PVDF
> filters, but we see it with both.
> 
I find that this happens with Amersham's ECL kit if the secondary antibody
is not washed long enough and there is still some reagent left on the blot
when you wrap it to expose to film.  The five washes after the secondary
are very important to dilute it out - any of the enzyme left in solution
will cause background problems.  I also take the blot and dry it with
KimWipes before putting it in the wrap, this makes a big difference as
well.

Regards,

Helmut



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