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
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: garav...@msu.edu
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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.
