Problems revealing western blots
Alexandre.BlaisNOSPAM at crchul.ulaval.ca
Mon Dec 4 21:06:51 EST 2000
This message is quite long but describes in detail a problem I am
experiencing with my western blots
I use Piece Supersignal West Dura in western blots experiments. It gives
the high sensitivity needed to detect a very weakly expressed protein.
have been using it for several months and it has always been perfect.
However, for the last three weeks, I am experiencing problems. The
chemiluminescent signal is very strong immediately after incubation of
nitrocellulose sheet with the product. The signal is so strong (as it
always been in past experiments) that I can see a brown precipitate on
nitro sheet, and I can actually see the light emited, in the dark room.
to 30 seconds exposal of the film is enough to obtain a strong signal.
These signs indicate me that the detection worked perfectly. However, 5
minutes after incubation with the SuperSignal reagent, the signal is no
longer detectable. Even a 60 minutes exposal will not give the slightest
signal on the film.
In order to find the problem, I tested a different secondary antibody,
the problem persists. So I tried a different primary antibody (directed
against the same protein but detecting a different epitope on that
from the same species and with the same secondary antibody. This
gives a strong signal that lasts for several hours. So the problem seems
arrise from my primary antibody. Also, on the western blot, I detect
some other bands (probably unspecific background bands) that give much
weaker signal and that do not produce the brownish precipitate. This
signal also vanishes prematurely. So the hypothesis that the HRP is
inactivated by too much "signal" does not hold. I tried a different
chemiluminescent reagent from another supplier (Renaissance, from NEN)
and the problem is the same.
So my questions are : how is it possible that the signal disappears so
quickly after incubation with the SuperSignal reagent ? And how could
problem be antibody-specific ? Is there some antigen-antibody
that are very labile under the conditions taking place in the
chemiluminescent reaction ? Pierce tech assistance could not find out
what is going wrong...
Thank you for the help or for any hint. I know that my problem is quite
particuliar, but if this problem is common to other , let me know what I
can do about it.
Again, thank you,
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