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. 2011 Nov 1;8(6):1158–1172. doi: 10.4161/rna.8.6.17699

Figure 6.

Figure 6

TAP purification of Nop19p-TAP under stringent conditions. (A) Nop19p-TAP was affinity purified under native conditions (see Materials and Methods). Nop19p-TAP-containing complexes bound to IgG were extensively washed with buffers containing 200 mM, 1 M or 2 M salt (KCl) concentration before Tev elution. Final purified samples were separated by SDS-polyacrylamide gel electrophoresis and observed after silver staining coloration. Samples were next subjected to mass spectrometry analysis. (B) Proteins specifically associated with Nop19p. Interactions identified by mass spectrometry analysis were individually verified. 3HA-tag versions of proteins of interest (Dhr2p, Utp25p, Dhr1p, Utp17p, Pwp2p, Utp22p, Rrp5p) were expressed in a NOP19::TAP background strain. Total extracts were immobilized on IgG and submitted to extensive washes with buffers containing 200 mM, 1 M or 2 M salt (KCl) before elution. Eluted proteins were analyzed by western blotting using PAP and HA antibodies.