GST Antibody (8-326)

Immuno-precipitation of GST-Shp2 SH2 domain (Product #87709) (Lane 2, 39kDa) was performed by mixing 2µg of purified fusion protein with1µg of mouse monoclonal antibody recognizing GST (Product # MA4-004) overnight on a rocking platform at 4°C. The immune-complex was then captured on 50µl Protein A/G Plus Agarose (Product #20423). Captured immune-complexes were then washed extensively and proteins eluted with 5X Reducing Sample Loading Dye (Product # 39000). Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel. IP efficiency was compared to a non-IP loading control consisting of 1ug GST-Shp2 SH2 domain (Lane1). Proteins were transferred to PVDF membrane and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. Membranes were then probed with a mouse monoclonal antibody recognizing GST (Product # MA4-004) at a dilution of 1:1000 overnight rotating at 4°C. Membranes were then washed in TBS/0.1% Tween-20 and probed with Clean-blot IP detection reagent (Product #21230) at a dilution of 1:2000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using Pierce Super Signal West Dura (Product #34075).

Western blot analysis of GST-Abl SH2 domain (Lane 1, 35kDa) (Product # 87702) and GST-Grb2 (Lane 2, 38kDa) SH2 domain (Product #87700) was performed by loading 2ug of each purified fusion protein onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/0.1%TBST for at least 1 hour. Membranes were then probed with a mouse monoclonal antibody recognizing GST clone 8-826 (Product # MA4-004) at a dilution of 1:1000 overnight at 4°C on a rocking platform. Membranes were then washed in TBS/0.1%Tween-20 and probed with a goat anti-mouse-HRP secondary antibody (Product #31430) at a dilution of 1:25000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using Pierce SuperSignal West Dura (Product #34075).