Rabbit anti his antibodies

In vitro pulldowns were performed as described previously.^{22 (link)} Briefly, C. elegans GTP- and GDP-binding GTPase isoform (QXXL and TXXN) expressing plasmids were transformed into BL21(DE3)pLysS (Fisher Scientific, Hampton, NH, USA) and protein expression was induced by 1 mM IPTG at 37°C. All His-tagged GTPase proteins were purified using His-Bind resin (Novagen, Darmstadt, Germany) according to the manufacturer's instructions. All of the buffers contained 2 mM MgCl₂ and 1 mM Tris(Hydroxypropyl)Phosphine (THP). The proteins were dialyzed against 10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2 mM MgCl₂ and 0.1 mM THP, and snap frozen at −80°C. GST and GST::GAP domain isoform containing plasmids were transformed into BL21(Gold) (Fisher Scientific) and their expression products purified and immobilized onto Glutathione Sepharose beads by standard methods. Immobilized GST fusion proteins (30 μg) were incubated with 10 μg of His-tagged GTPase proteins in a buffer containing 10 mM Tris-HCl (pH 7.9), 100 mM NaCl, 2 mM MgCl₂ and 1 mM THP, 0.5% NP-40 and 10% glycerol. The incubation was carried out for 2 h at 4°C with agitation. The beads were washed in the same buffer three times and the proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). His-tagged proteins were immunoblotted with rabbit anti-His antibodies (Santa Cruz, Santa Cruz, CA, USA; 1 : 1000).