Mouse anti ha monoclonal antibody clone 2 2

The procedure of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) was described previously (38 (link)). Briefly, the culture medium of pDisplay-HA-BAFFL transfected 293T cells was mixed with 5% glycerol and 0.01% Ponceau S, then applied on 10% native PAGE gel. Mouse IgG (~160kDa), bovine serum albumin (BSA, 66kDa) and ovalbumin (OVA, 43 kDa) were electrophoresed as size markers. The separated proteins were transferred to a nitrocellulose membrane by conventional semi-dry blotting. The HA-tagged lamprey BAFF-like protein was reacted with mouse anti-HA monoclonal antibody (clone 2-2.2.14, Thermo Fisher Scientific). After washing with PBST (PBS containing 0.1% Tween 20), the blot was reacted with horseradish peroxidase (HRP) conjugating goat anti-mouse IgG (Southern Biotech). After washing with PBST, the HA-BAFFL was detected using SuperSignal West Pico (Thermo Fisher Scientific) on an Image station 4000 MM pro (Kodak).

To detect ligand-receptor interaction, 293T cells transfected with the pDisplay-FLAG-BCMAL receptor construct were incubated with HA-BAFFL containing DMEM for 30 minutes. After washing with PBS, the cells were reacted with a rabbit anti-FLAG polyclonal antibody (Sigma) and a mouse anti-HA monoclonal antibody (clone 2-2.2.14, Thermo Fisher Scientific). PE-conjugated goat anti-mouse IgG (Southern biotech) and Alexa 488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific) were used for detection. The stained cells were analyzed using a BD Accuri C6 cytometer and Cytobank (https://community.cytobank.org/).