Purification of inclusion bodies for antibody production
This protocol is extracted from research article:
Functional insights from a surface antigen mRNA-bound proteome
eLife, Mar 30, 2021; DOI: 10.7554/eLife.68136

For large-scale purification of inclusion bodies, 1 l of an IPTG-induced culture was grown at 27°C until reaching an OD600 0.8 and harvested by centrifugation at 5000 rpm for 20 min. The pellet was washed once with 1× PBS and stored at −80°C until further processing. Lysis of the bacteria was performed by addition of 500 µl of bacterial lysis buffer and 50 µl of lysozyme solution (see above). Furthermore, the samples were mixed by vortexing for 3 s, incubated for 4 hr at 4°C with constant rotation, and subsequently subjected to three freeze/thaw cycles using liquid nitrogen. Inclusion bodies were pelleted by 20 min centrifugation at 15,000 rpm and 4°C, washed once with bacterial lysis buffer containing 1% Triton X-100, and eventually resuspended in 10 ml of 8 M urea. These were then left to dissolve for 16 hr at 4°C with constant rotation.

After determining the protein concentration spectrophotometrically (A280; Nanodrop, Thermo Fisher Scientific, Karlsruhe, Germany), 0.5 mg of the protein in inclusion bodies was then separated by SDS-PAGE and the band corresponding to NusA-CFB2 was cut from the gel. The gel slice was submitted to David’s Biotechnology (Regensburg, Germany) for the generation of antisera in rabbit.

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