Figure 1. NIH/3T3 cell lysates (500 µl at 1 mg/ml) were treated <i>in vitro</i> with GTPγS or GDP to activate or inactivate Ras (refer to optional step C in protocol). The lysates were then incubated with glutathione resin and GST-Raf1-RBD (lanes 2 and 3). GTPγS-treated lysate was also incubated without GST-Raf1-RBD in the presence of glutathione resin as a negative control (lane 4). Western blot analysis of cell lysate (20 µg, lane 1) or 20 µl of the eluted samples (lanes 2, 3, and 4) was performed using a Ras mouse mAb. Anti-mouse IgG, HRP-linked Antibody #7076 was used as the secondary antibody.
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