发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: 怎样调整gel，走好SDS-PAGE?
发信站: The unknown SPACE (Fri Oct 11 14:53:52 2002), 站内信件

throw in a couple of points:
when you run an SDS-PAGE, you try to fractinate the protein
by the matrix of acrylamide. so the qualities of both the
gel and the samples are important to determine migration.
basically, do not use gels that have been stored for a long
time, use fresh AP and high-quality of acrylamide to make a
PAGE gel, giving long time for the bottom separation gel to
polymerize; or, just buy a commercial gel from BioRad, either
non-gradient or gradient (this one seems to be better for
fractionation). however, do not use VWR stuff when you try
to dry the gel after running radiolabeled samples, since the
gels tend to crack.
2nd, for the samples, use good loading buffer. do not add too
much SDS (it is used in RIPA buffer, TRIdetergent lysis),
which will cause problem. it helps a lot if you desalt and
concentrate the samples first. try to avoid existence of genomic
DNA in the samples, so the previous isolation step is really
important. once the protein is in loading buffer, when i reuse
it, i would just warm it in hot water (not boil) for a couple
of minutes and spin @ 10000g 2min. while loading, as others
have mentioned, try to load equal amount, supply the blank
wells with loading buffer. wash the tank, and use fresh running
buffer. it is better to check that the electrodes are fine,
particularly the wires are straight. typically, run the gel
@ constant Amp, e.g., 30mA for ea. mini-gel. but i would rather
run @ constant voltage first, let the gel run somewhat faster
to minimize sidewalk.

【 在 sianna (交流) 的大作中提到: 】
: 我的胶走得歪歪扭扭的，不只是什么原因，经常出现下列情况：
: ~，或者(),即两侧的带都向边上弯曲，
: 如果是胶的原因，应该怎样调整？


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※ 修改:．Marble 于 Oct 11 15:05:49 修改本文．[FROM: 146.186.]
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 146.186.]