what the inclusion body pellets look like

S.Ballal ballal_sv at hotmail.com
Thu Dec 19 22:43:04 EST 2002


I guess Artem has given a description which cannot be improved upon.
Normally if you are getting a great yield you can get upto few grams
of IB pellet from a liter of broth ( If you are from research lab then
few 100 mg per 100 ml of culture of OD between 10-15).  One can see
the IBs under the phase contrast microscope while they are still in
the cells with 1000x magnification without any staining.

The jelly like stuff is most likely to be DNA or similar material
which can be taken care of by simply continuing your lysis for little
longer (to break the DNA in small and less viscous material) or
washing your pellet with Tris buffer by resuspending and
centrifugation several times. A 1-2% Triton-X 100 wash also gives a
good result.

Let me know if you need any more input.

Best of luck

S.Ballal
Indus Biotherapeutics Ltd.
Ahmedabad INDIA
http://www.indusbio.co.in



"Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote in message news:<cZ5M9.127189$%p6.14061241 at twister.neo.rr.com>...
> Most of the time dense inclusion bodies look kind of like very very fine
> yellowish-white powder. Inclusion bodies should be soluble in certain
> detergents and in strong denaturants. Resuspension of IB in simple buffers
> usually results in clear buffer and IB pellet after centrifugation.
> Various semi-folded forms of proteins can result in the effect(s) that you
> describe. Some DNA-binding proteins also create a 'jelly' after lysis due to
> crosslinking of DNA framgents.
> 
> A.G.E.
> <fxiong at asu.edu> wrote in message
> news:20021218065517.24337.qmail at ww02.hostica.com...
> > Hi, all,
> >
> > I recently extracted overexpressed protein which was expressed probably in
> the form of inclusion body. I break the E. coli cells and spin down the
> pellet. It seemed kinds of difficulty to resuspend the pellet. Also, the
> suspensions are pretty jelly-like (Viscosity problem?) I'm wondering is this
> normal for inclusion body isolation?
> >
> >
> >
> http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0



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