Please help with 2D gels
jfackent at bio.indiana.edu
Sat May 22 12:16:01 EST 1993
Maybe someone can help us. We've been running 2D gels of labeled testis
proteins from Drosophila for quite a few years. For the last couple of months
we've been seeing problems that we've never seen before, problems that just
won't go away. We prepare samples by boiling dissected testes in SDS sample
buffer with BME. Our first dimension "worms" contain NP40, acrylamide, urea,
and LKB ampholines (which always worked before. Sometimes we use 1:1:1:1
3.5-10:4-6:5-8:7-9, but often we'll change the ratios to get an expanded
range gel). Then we run a standard 2nd dimension gel. The problem is in the
tubulin portion of the gel, which is the only part we're interested in. All
other spots in all other portions of the gel are consistently perfect. The
tubulin problem (about 50 kd, pI is between 5.5 and 6.5) takes one of two
forms: first, the beta-tubulin spot splits severely; part of it accumulates
just below the alpha-tubulin spot, and the rest accumulates at its own, more
acidic position. A streak connects these spots. Second, the beta-tubulin spot
splits, but not as severely. That is, the entire beta-tubulin pool migrates
to its proper position, but the spot splits, generating a "peanut" shape.
On the surface, it looks like a solubility or dissociation problem.
Anybody have any ideas? Similar experiences?
Please feel free to respond by e-mail to jfackent at ucs.indiana.edu.
I'd really appreciate any suggestions.
Thanks a lot, Jim Fackenthal
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