NOS western blot problem

Anna Durrans anna.durrans at physiol.ox.ac.uk
Fri Jul 13 08:39:40 EST 2001


Hi everyone,

I have been running westerns for eNOS, nNOS and iNOS successfully using
various rodent organ tissues. I use actin as a housekeeper.

In order to show reproducibility I recently re-ran the exact same lysates
again, along with some freshly made lysates. Strangely I found that the
actin signal was very much weaker in some (not all) of the original
lysates,
as wells as in the fresh lysates.

I re-ran all these again, and while there is a pretty stong actin signal,
it
is still weaker in those tissues which before showed a weak actin signal.

I am not sure what could have happened. The NOS signal in ALL the tissues
was fine - so it is not the lysates themselves but possibly the actin
specifically which has degraded.

Has anyone else had this problem? Does anyone know what could have
happened?

I would be really grateful for any suggestions.

Anna



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