Fig. 5. Identification of p40. (A) Immunoaffinity purification of p40 associated with R4op. Large-volume RRL translation reactions were incubated with R4op mRNA (lanes 1 and 2) or without (lanes 3 and 4). R4op-containing complexes were affinity-purified using anti-opsin antibody beads and proteins released from R4op by elution with 0.1% Triton X-100 (TX) (lanes 1 and 3). Remaining bound proteins were eluted from the column by using an acidic glycine buffer (gly) (lanes 2 and 4). Eluted proteins were separated by SDS-PAGE and silver stained. The protein band of about 40 kDa was cut out, proteins were eluted and peptide sequences determined by mass spectroscopy. Peptide sequences identified p40 as Asna1. (B) Immunoprecipitation of R4op x p40 crosslinked product. R4op was synthesised in RRL and aliquots of the reaction were either crosslinked with BMH (+) or incubated with DMSO solvent alone (–). Aliquots of both reactions were either directly applied to the gel (lanes 1 and 2) or immunoprecipitated by anti-opsin antibody (lanes 3 and 4), an anti-Asna1 antibody (lanes 5 and 6) or a pre-immune serum (lanes 7 and 8) and characterised by SDS-PAGE and autoradiography.