Ipkine hrp goat antimouse igg hcs

Co-immunoprecipitation (co-IP) was performed as previously described (Yan et al., 2017 (link)). The cells were washed once with cold PBS, lysed in lysis buffer for IP (Beyotime, #P0013) containing a protease inhibitor cocktail (Roche) at 4°C for 1 h, and then centrifuged at 12,000 g for 15 min at 4°C. After centrifugation, a small fraction of the supernatant was frozen for subsequent analysis (cell extract) as the input. The remaining fraction was incubated at 4°C with 1 μg of the desired antibody on a rotator overnight. Then, the mixtures were incubated with Protein A/G agarose beads (Abcam, #ab193262) with rotation at 4°C for 6 h. After the incubation, the beads were washed three times with wash buffer, boiled in loading buffer at 95°C, and then subjected to western blotting. The following specific secondary antibodies were used to avoid overlap of the bands for the target proteins with those of the immunoglobulin light chain and heavy chain: IPKine HRP Goat AntiMouse IgG HCS (Abbkine, #A25112, 1:5,000), IPKine HRP Goat AntiRabbit IgG HCS (Abbkine, #A25222, 1:5,000), IPKine HRP AffiniPure Goat AntiMouse IgG Light Chain (Abbkine, #A25012, 1:5,000), and IPKine HRP AffiniPure Mouse AntiRabbit IgG Light Chain (Abbkine, #A25022, 1:5,000).

For CO‐IP, VpPR10.1 and VpVDAC3 were cloned into p35S, GFP (VpPR10.1‐GFP) and p35S, 3Flag (3Flag‐VpVDAC3) vector. Agrobacterium strain GV3101 containing the constructs was infiltrated into N. benthamiana leaves. 0.7 g flash‐frozen homogenized N. benthamiana leaf tissue was suspended in 1.5 mL IP buffer (Choi et al., 2012), followed by incubation with monoclonal anti‐GFP (ABclonal) overnight at 4 °C with gentle shaking. The immune complexes were incubated with prewashed monoclonal anti‐GFP agarose (Sigma‐Aldrich) for 5–6 h at 4 °C and were centrifuged and washed with immunoprecipitation buffer (Choi et al., 2012). Resuspend proteins were separated by 10% SDS‐PAGE. Anti‐GFP mouse monoclonal antibody (ABclonal) and flag‐specific monoclonal antibodies (Santa Cruz) were both probed at 1,2000 dilution to detect goal proteins. IPKine™ HRP Goat Anti‐Mouse IgG HCS (http,//www.abbkine.com/) was used as a secondary antibody.