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Expression in E.coli

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16 replies to this topic

#16 ram



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Posted 12 September 2010 - 08:14 AM

This is how I do it:

harvest 100 ml culture
re-suspend pellet in 5ml lysis buffer, sonicate
supernatant: soluble fraction
pellet: re-suspend in 500 ul lysis buffer: insoluble fraction
Use 1-2 ul of the insoluble fraction for loading on PAGE

I add regular PAGE loading buffer (which contains DTT, a protein denaturant) to all (soluble/insoluble) samples and heat them at 100C for 2 min.
The combinatorial effect of DTT and heating denatures and solubalise the protein/s in inclusion bodies
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#17 bhavana



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Posted 12 September 2010 - 10:56 PM

k on what basis u r deciding the amount of lysis buffer to be added for 100ml of culture.
And dont u add chaotropic agents like urea, etc to the insoluble fraction?
500ul of which lysis buffer u use to add to the insoluble fraction,
whats ur lysis buffer composition?

Edited by bhavana, 12 September 2010 - 11:00 PM.

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