INCLUSION BODIES
Frequently proteins produced in E. coli or other bacterial hosts collect in insoluble inclusion bodies. Though highly immunogenic, inclusion bodies frequently contain endogenous E. coli proteins in addition to the overexpressed protein of interest. For this reason subtractive chromatography (using immobilized E. coli proteins) is recommended in order to remove the undesired antibodies from the immune serum.
Preparation of Inclusion Bodies for Antisera Production*
- Wash inclusion body in 1% NP-40, 100 mM NaCl, 1 mM EDTA, 50 mM Tris , pH 8.0
- Centrifuge at 10,000 Xg for 10 minutes
- Wash the pellet with 100 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 8.0
- Centrifuge at 10,000 Xg for 10 minutes
- Freeze pellet at -70 and ship on dry ice
* Adapted from Harlow & Lane. Antibodies a Laboratory Manual (1988).