Anti ha high affinity monoclonal antibody clone 3f10

GTP-bound Rho proteins were analyzed as described (Calonge et al., 2003 (link)) using a Rho-GTP pull-down assay. Briefly, cell extracts from wild-type or rga6Δ cells expressing HA-cdc42⁺ or HA-rho2⁺ from their own promoter were obtained by mechanical breakage of the cells using glass beads and FastPrep. Cells were resuspended in 500 μl of lysis buffer (50 mM Tris-HCl, pH 7.5, 20 mM NaCl, 0.5% NP-40, 10% glycerol; 0,1 mM dithiothreitol, 1 mM NaF, and 2 mM MgCl₂ containing 100 μM p-aminophenyl methanesulfonyl fluoride, leupeptin, and aprotinin). A 10-μg amount of either GST-CRIB or GST-RBD, previously obtained from E. coli DNA expression, purified, and coupled to glutathione–Sepharose beads, was used to precipitate the GTP-bound Rho GTPases from 2 mg of the total cell lysates. The extracts were incubated with the beads for 2 h at 4°C, washed four times, and blotted against anti-HA high-affinity monoclonal antibody (Clone 3F10; Roche Molecular Biochemicals, Mannheim, Germany) to detect the corresponding GTP-bound HA-Rho protein. The total amounts of Rho proteins from the extracts were determined by Western blot using the anti-HA monoclonal antibody.