发信人: Cathleen (蛹-@-化蝶), 信区: Biology
标 题: Re: How to do a western on insoluble protein
发信站: Unknown Space - 未名空间 (Mon May 10 01:18:45 2004) WWW-POST

the more harsh your solution, the more components will disolve, of course the
more you will see on the gel.
If you want to disolve all the pellet, try the most "harsh" solution you can
imagine, like 5%-Triton-2%SDS-8M urea.....

【 在 worryless (无忧) 的大作中提到: 】
: I have fibroblast cells.
: After I lyse them in lysis buffer containing 1% Triton X-100 or 1% NP40,
: (both with 50mM pH7.4 Tris, 150mM NaCl)
: I centrifuge them at 14000RPM at 4C.
: The supernatant I get is the Triton X-100 (or NP40) soluble fraction,
: the pellet on the bottom of the eppendorf tube is Triton X-100 (or NP40)
: INSOLUBLE fraction.
:
: If I want to run a SBS-PAGE and then do a western blot to see what is in the
: INSOLUBLE fraction, can I just resuspend the INSOLUBLE fraction in 1XSDS
: sample loading buffer and run the gel?
: Or do I have to lyse the INSOLUBLE fraction in a more harsh lysis buffer
: (say lsysi buffer plus 0.2%SDS), and then add 2XSDS sample loading buffer
: and then run the gel?
:
: thanks for any suggestions or comments!
:
:
:

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