western blot problems
duriya at excite.com
Sat Dec 30 08:39:39 EST 2000
I used western blot methods to detected the trans-gene protein which
expressed in transgenic plants. From the literature, the detectable level of
trans-gene in plant is 50 micrograms. In order to obtain that amount, I had
to use thick SDS gels (about 2 mm) for western bloting. Then I faced the
problem of incomplete blotting by using semi-dry blot. (Before I changed to
thick SDS gels, I used to blot with thin SDS gels (about 1 mm) which gave a
complete blotting.) I had heard that the wet bloting(submerge) could solve
this problem. So I deciced to use wet blot, but I still found the same
problems in bloting and I also found new problems, which were a non-specific
bands in the purified protein lane that usually uses as the positive
control. For the protein total protein from plant that does not express the
protein ( I usually use as the negative control), it gave a signal that
never found before when using thin-gel bloting.
I need to know why the wet blot give non-specific signal bands. I
aliquat the positive and negative control protein and I use it all every
time I did western, so I did't think it was a degraded product. My transfer
buffer composes of Tris, Glycine and methanol.
Thank in advance.I only need to detect the protein expression in plants
but the result are not consistant. I have no idea what I have to do next.
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