Western blot analysis of GST-Abl SH2 domain (Lane 1, 35kDa) and GST-Grb2 SH2 domain (Lane 2, 38kDa). 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 GST antibody [8-326] at a dilution of 1:1000 overnight at 4ºC on a rocking platform. Membranes were then washed in TBS/0.1%Tween-20 and probed with a HRP-conjugated secondary antibody. Membranes were washed and chemiluminescent detection was performed.

Immuno-precipitation of GST-Shp2 SH2 domain (Lane 2, 39kDa) was performed by mixing 2μg of purified fusion protein with 1μg of GST antibody [8-326] overnight on a rocking platform at 4ºC. The immune-complex was then captured on 50μl Protein A/G Agarose. Captured immune-complexes were then washed extensively and proteins eluted. 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 GST antibody [8-326] at a dilution of 1:1000 overnight rotating at 4ºC. Membranes were then washed in TBS/0.1% Tween-20 and probed with a proper secondary antibody. Membranes were washed and chemiluminescent detection was performed.