Specific V5-tagged proteins synthesised by TNTH were precipitated from 5-10 ml of reaction product using the anti-V5 monoclonal antibody (Invitrogen) at the recommended dilution in 600 ml of 16 RIPA buffer (diluted from 10x stock: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.15 mM NaCl, 0.1% SDS, 0.5% Empigen BB, 0.1 mM phenylmethylsulphonylfluoride) for 1 hr at 37uC. This was followed by a second incubation of tube for 2 hr rotating at room temperature with goat anti-mouse immunoglobulin G agarose beads (Sigma) to absorb the immune complexes. The beads were washed three times with 500 ml 16 RIPA buffer and once with 500 ml PBS. The beads were resuspended in sample buffer for analysis by SDS-PAGE and autoradiography as before.