Anti groel antibody

Profiling of OMPs was performed as described previously (11 (link)). Briefly, after bacterial cells were disrupted by sonication, the OM was harvested by ultracentrifugation, washed with Tris buffer containing 2% N-lauroylsarcosine, and purified by ultracentrifugation again. OMPs were extracted from the purified OM and separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were identified by nano-flow liquid chromatography coupled to tandem mass spectrometry. Immunoblotting was performed using anti-OmpK35 antiserum and anti-GroEL antibody (Abcam).

E. coli BL21(DE3) cells were cultivated at 37°C in LB medium containing ampicillin (50 µg/mL). Protein expression was induced with 0.5 mM IPTG when the cultures reached an OD₆₀₀ of 0.5, and the cells were incubated for an additional 6 h. The cells were harvested by centrifugation at 16,300×g for 10 min and then disrupted by sonication on ice.
The protein expression was analyzed by SDS-PAGE and western blotting. Aliquots of cell lysates were electrophoresed on 12% SDS-polyacrylamide gels and electro-transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were hybridized with an anti-GFP mouse antibody (Sigma-Aldrich, St. Louis, MO, USA) and an anti-groEL antibody as the internal standard (Abcam, Cambridge, MA, USA), followed by an HRP-conjugated anti-mouse IgG goat antibody (Bio-Rad, Hercules, CA, USA) prepared in TBST buffer (20 mM Tris-HCl, 100 mM NaCl, and 0.1% Tween-20, pH 7.5) containing 5% skimmed milk. The hybridized bands were identified by colorimetric detection using an Opti-4CN substrate kit (Bio-Rad).

Cells were lysed using a one-step bacterial active protein extraction kit (Sangon Biotech, China). The lysates were incubated with anti-GroEL antibody (Abcam, USA) at room temperature for 1 h. Protein A/G Agarose Resin 4FF (Yeasen, China) was extensively washed with washing buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0) and centrifuged at 500 rpm for 1 min. The lysate-antibody mixture was subsequently added to the washed beads, followed by incubation for 30 min at room temperature. The beads were then washed with washing buffer three times and eluted using 25 μL 1× SDS PAGE loading buffer³³ .