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Need HeLa cell extract prepartion protocol


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#1 biowei

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Posted 04 February 2005 - 08:10 PM

HI,everyone
Could you give me a protocol for Hela cell extract preparation which amenable for further GST pulldown assay?or from your viewpoints,whether it is a good way for screening binding partner?your kind help will be greatly appreciated.


Best regards


biowei

#2 Sprag

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Posted 08 February 2005 - 09:56 AM

There are many different lysis buffers for mammalian cells dependent on which proteins you are interested in.
1% Triton X-100 in 50 mM Tris (pH 7.5), 150 mM NaCl + protease inhibitors is the most basic one.
Alternatively you can use 0.5% NP-40 in 50 mM Tris (pH 7.5), 150 mM NaCl, 10% Glycerol + protease inhibitors..

The method is widely used for identifing new binding partners, but it is VERY important to get your GST-fusion protein to very high purity, so you can be confident that what you pull out is truely binding partners and not some left overs from the purification. Always use GST alone as a control. Of course these interactions have to be verified by other methods before you can truely believe it.

have fun...




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