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 18:15:30 2004) WWW-POST


【 在 worryless (无忧) 的大作中提到: 】
: Thank you for your reply.
: I understand that harsher lysis buffer will lyse it all,
: but the problem is, I don't know whether too harsh lysis buffer will
interfere
: with the SDS-PAGE.
: That's why I ask whether 0.2%SDS+1%NP40(50mMTris and 150NaCl) will be enough
~~~~~~~~~~~~~~~~~~~~~~~~ this is not harsh to
disolve
all proteins, like some membrane proteins, they are really hard to disolve.

High concentration of TritonX-100 will affect SDS-PAGE, you can try 8M urea,
or you can even add 8M urea when casting the SDS-Gel, search methods with key
words like" disolve membrane proteins", "membrane protein SDS-PAGE", you'll
get more informative stuff.

Good luck



: to lyse the Triton X-100 or 1% NP40 INSOLUBLE fraction and disolve all the
: proteins completely.
:
: 【 在 Cathleen (蛹-@-化蝶) 的大作中提到: 】
: : 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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