ief gels: samples jumping lanes?
will.fuller at kcl.ac.uk
Thu Feb 10 13:59:43 EST 2000
I wonder if anyone can throw any light on a problem I'm having
interpreting Western Blots from ief gels.
Briefly, my gels (BioRad mini-Protean II system) are composed of 9.2M
urea, 2% (each) of TX-100, nonidet P40 and CHAPS, 2% ampholytes, and 4%
acrylamide. Gel loading buffer is the same except 9.5M urea and no
acrylamide. I add 50ul of sample (homogenate) to 200ul loading buffer.
Transfer is semi-dry in 0.7% acetic acid for 2.5 hours at 100mA per
mini-gel. I can stain my gels after transfer and my PVDF membranes with
Coomassie after probing.
Generally there is some protein left in the gels following transfer, and
it usually looks good, that is to say it runs evenly and there is
protein in the lanes I've loaded and none in the lanes I haven't. The
same is true when I stain my pvdf membrane after probing. However, the
images I get following probing the blots are very confusing. It appears
that my samples are able to jump from lane to lane. That is, where I
load a "blank" lane (that is, my loading buffer only) I get a signal.
This signal seems to be related to the signals from the lanes either
side of the blank, and is of comparable magnitude. I use a fresh loading
tip for each well when loading. The "blank" loading buffer is not
contaminted with protein (I have made it up fresh many times), and of
course there is no sign of any protein in these blank lanes on my
stained membranes and transferred gels. What's more I don't believe this
is a case of lateral diffusion in the gel during running because the
signals from the blank lanes are always "discrete", that is there is
always a gap between the signal from the blank lane and that from the
lanes alongside it.
I am not loading a large volume of sample for the size of the well (5ul
in a well that will hold 15ul), and pay particular attention to ensuring
that when the gels are loaded they are not moved or disturbed in a way
that might allow sample to "jump" from one lane to the next. When I put
the film down to get an image from the membranes after probing I am
certain there is no lateral movement of the film that might create some
sort of "ghost" image.... I do quite long exposures (about 1-5 minutes)
in a film cassette.
I can see no explanation for this phenomenon.... There is no protein
there according to Coomassie stains, but there is a similar amount of
the protein I am blotting for in blank and sample lanes according to
Western analysis. I routinely run SDS PAGE gels and blot them without
this sort of problem, so it seems to be specific to ief gels.
If anyone has any tips, experience, and so on I'd be delighted to hear
from you. This has been going on for some time now and I'm heartily sick
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