Protein Co-Immunoprecipitation from E. coli

Cas2-Flag on pET-28a, FtsZ-HA on pET-11a and the vector controls were co-transformed into E. coli BL21-AI.^{16 (link)} Stable transformants selected on LB plates with kanamycin (50 μg/ml) and ampicillin (100 μg/ml) were cultured in LB medium with the two antibiotics plus 0.2% (wt/vol) L-arabinose and 0.2 mM IPTG at 37 °C for 3 h. Cells were pelleted by centrifugation at 4000 rpm for 5 min and subjected to a standard ultra-sonication protocol described in an early publication.^{26 (link)} The supernatants were collected after a 10 min centrifugation at 12,000 rpm, and the same protocol was repeated 2 times. The lysates without debris were subjected to an immunoprecipitation assay^{23 (link)} with anti-HA beads (Millipore catalogue number IP 0010) for FtsZ and anti-Flag beads (Millipore catalogue number FLAGIPT1) for Cas2. The total proteins eluted from the beads were separated on 4–12% Bis-Tris Plus gels (Invitrogen catalogue number NW04122BOX) with MES SDS running buffer (Invitrogen catalogue number NP0002); transferred onto a PVDF membrane (Invitrogen catalogue number IB24001) by iBlot II (Invitrogen); and subjected to standard western analysis with an HRP-conjugated anti-Flag antibody (Sigma-Aldrich catalogue number A8592-1MG) and HRP-conjugated anti-HA antibody (Sigma-Aldrich catalogue number H6533).