Problems with Westerns
Philip L. Carl
plc at med.unc.edu
Thu Jul 29 11:51:19 EST 1993
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.
On a related front, we have tried stripping antibodies
off nitrocellulose blots with elevated temperatures and SDS
following published protocols and then reprobing with
different antibodies. It works fine, but when we try this
with PVDF membranes the blotted proteins come off too. Has
anyone a method for stripping of such blots and reuse?
Finally, we have seen one more rather bizarre problem.
If we load small amounts of protein things may work OK, but
when we load larger amounts the signal, instead of getting
greater disappears. I assume that this may reflect a very
high concentration of the antigen at the gel band, such that
secondary antibodies are unable to "find" the primary
antibody. Transfer is fine as is confirmed by staining. Ha
Have any others encountered these sorts of problems who can
offer solutions? Please answer via the news or email to
plc at med.unc.edu. Thanks.
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