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agree with Michael, use low concentration of detergent. I normally resuspend the cell in buffer and sonicate, spin down at 6000 g., resuspend again with buffer containing small amount of detergent and resonicate, spin down again at 6000 g. finally was with buffer again (resuspend and spin down).

By the way, Michael, I've tried washing with 2 M urea but found some protein in the wash out (soluble), very small amount (not detectable by SDS PAGE but faint band observed upon WB). Is it a sign that the protein was properly folded like you mentioned?

Wangsa

2009/10/28 R.M. Garavito <[log in to unmask]>
As Artem and Ezra have already mentioned the basic aspects of isolating IBs, I should point out that most people wash the pellet of the first low-speed centrifugation 2-3 more times.  While Ezra suggests using increasing amounts of urea, you may have to tailor the composition of the wash buffer to your unique protein.  For example, many wash with a low concentrations of detergent (0.1 % Triton X-100, Tween-20, or any other detergent) or varying amounts of salts.  The washed pellets tend to get whiter and whiter as the IBs get cleaner and cleaner.

There are reports that IBs of some proteins, occasionally membrane proteins (e.g., mitochondrial carriers: see Ron Kaplan's work with the citrate carrier), will form properly folded protein in IBs when the E. coli cultures are grown at low temperature (12 - 20C).  So don't immediately assume your IBs are misfolded protein; you may be lucky and just need to find the right solubilization method (Jevsevar et al., Biotechnol. Prog. 2005, 21, 632-639).

Michael


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R. Michael Garavito, Ph.D.

Professor of Biochemistry & Molecular Biology

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Michigan State University      

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On Oct 28, 2009, at 7:44 AM, Ezra Peisach wrote:

Artem has already responded - but I believe you will pull down the cellular debris with the IB.
In my prior experience, post centrifugation, you can wash the IB/debris with increasing amounts of urea in you solubilization - and you may find that the other cellular debris stays in solution before the IB goes in. (1M, 2M, 4M urea....) - and after recentrifugation, I found that does a pretty good job of cleaning up the media.

Ezra


megha goyal wrote:


Dear All,
Our protein is expressed as inclusion bodies and I want to separate inclusion bodies from E.coli from the cellular debris after* *lysis of the cells by sonication.

Can I do this by normal centrifugation? and if yes, at what speed?

Our centrifuge has maximum speed of 14000 rpm. Can we do the separation using this centrifuge and if so how.


Thanking in anticipation.






--
Wangsa Tirta Ismaya
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Utrecht, The Netherlands