Problems revealing western blots
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Tue Dec 5 09:40:30 EST 2000
Alex <Alexandre.BlaisNOSPAM at crchul.ulaval.ca> wrote:
: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.
I think the formal answer is obvious. If the signal is so strong
that you can actually see the precipitate, then you are using way
too much primary antibody. Dilute it further ten times (or whatever
it takes) and the problem _will_ diasappear.
As far as why this is happening, it's hard to tell because because
composition and exact properties of Pierce's Supersignal ECL are
unknown, and I am not sure what aspect of it makes it emit
light for a lot longer. Here is my best bet:
AFAIR, luminescence is fast (unlike, say, phosphorescence) -
the molecule emits light fast and does not anymore. If so, the
longevity of Pierce's signal probably comes from the fact that it
is a lot "stronger" (better quantum yeild, blue light provides for
better detection, etc). If so, you need less molecules to achieve
the same sensitivity. Now, imagine that insoluble product
precipitate inhibits HRP (probably just physically obscuring
it from substrates, but not necessarily). That means that
reaction that produces less precipitate by virtue of having
smaller substrate concentration will keep producing light
longer - as long as enzyme is active and there is enough
substrates. If this fantasy of mine is correct, then your signal
disappear quickly because you have so much HRP bound
to the site that it makes so much precipitate that it kills HRP.
Premature faiding of your minor bands be explained in the
above scenario by depletion of substrates by excess of
enzyme in other parts of the blot. Just a guess...
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