antibodies prep. from inclusion bodies

Tony Hodge tph at mrc-lmb.cam.ac.uk
Thu Jul 27 09:24:11 EST 1995


In article <jpl-270795132513 at 193.51.160.15> , jpl at ibcp.fr writes:
>Subject: antibodies prep. from inclusion bodies
>From: jpl
>Date: Thu, 27 Jul 1995 13:25:13 +0200
>> I've got an insoluble recombinant protein from E. coli and I'm looking for
>protocols to prepare antibodies from inclusion bodies. Please help me!
>

 Patricia

There area number of ways you could proceed.  Proteins in inclusion
bodies are solubilised with Urea (6-8Molar), Guanidine or
ionic/noninoic detergents (SDS, sarkosyl etc - see Huang et al 1993,
BioTechniques Vol 15, No 6 pp989-992; or Frangioni&Neel 1993,
Analytical Biochem. 219,pp179-187).

Once solubilised you may be able to purify your protein further by
various forms of column chromatography or affinity chromatography
etc before inoculating.  Another method that can be used if the
protein is relatively pure when solubilised and that is to separate
out on a preparative PAGE gel then electro elute (eg in
Schleicher&Schuell Biotrap BT100) and inject.  Also you could blot
from the gel, grind up the membrane and inject that ore even grind
up the PAGE gel. These later two methods seem to work quite well at
producing an immune response but the question is always 'will the
resultant Ab recognise the native protein?' Check out 'Antibodies -
a laboratory manual Eds Harlow and Lane from the Cold Spring Harbour
Lab  1988 and chapter 5 (like maniatis but for Abs!) here they also
say (page 91) that you can inject the inclusion bodies direct after
sonication!

I've had luck with solubilisation in SDS, PAGE gel, electro-elution
and then immunisation but a colleague had problems with the
electro-elution stage so the yield here may be protein dependent.

Hope this helps, do Email me if you need specifics.


_____________________________________________________
Tony P Hodge
Structural Studies Division
MRC-LMB,  Hills Road 
Cambridge,  CB2  2QH,  UK
Tel (01223) 402260
Fax (01223) 213556
tph at mrc-lmb.cam.ac.uk
http://131.111.84.120/tph/HomePage.html
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