Gfp monoclonal antibody

Prior to each pull‐down assay, the Glutathione Sepharose 4B resin (GE Healthcare) was incubated with 5% BSA for 5 min and washed three times with 500 μl GST pull‐down buffer containing 50 mM Tris–HCl pH 7.0, 1 mM DTT, 50 mM KCl, and 5% glycerol. One‐hundred microliter of 2 μM Glutathione Sepharose 4B attached GST SPIN90 full length was mixed with 250 nM Arp2/3. A concentration gradient (0, 0.5, 2, and 4 μM) of GMF or GFP‐VCA was mixed with the loaded resin and allow to incubate for 1 h at 4°C. The resin was then washed three times with 300 μl of GST pull‐down buffer. Resin‐attached proteins were eluted with 50 μl of 20 mM GSH. The sample was separated by SDS–PAGE for western blot analysis. Anti‐ArpC2 (Sigma, HPA008352, Rabbit) was used to detect Arp2/3. GFP Monoclonal Antibody (Invitrogen, A‐11120, Mouse) was used to detect the GFP‐VCA.
The negative control experiment was done in the same way except 50 μl of 2 μM Glutathione Sepharose™ 4B attached GST was mixed with 250 nM Arp2/3, with or without 4 μM GMF or GFP‐VCA for 1 h at 4°C.