Identification of p47(1-170)-binding proteins

His-tagged p47(1-170 amino acid) (100 μg) was biotinylated using EZ-Link Sulfo-NHS-LC-biotin (Pierce), bound to immobilized Streptavidin beads (Sigma), and washed with 1 M KCl in buffer (20 mM Hepes, 1 mM MgCl2, 1 mM DTT, 0.5 % Triton X-100, 5 % glycerol, pH 7.4).
Rat liver tissues were homogenized in a five-fold volume (w/v) of buffer (0.1M KCl, 30mM Tris-Cl, 1 mM MgCl2, 250 mM sucrose, protease inhibitor cocktail [Roche], pH 7.4) with a Potter-Elvehjem homogenizer using two different Teflon pestles (clearance of 0.35 and 0.05 mm). The homogenate was centrifuged at 2,000g for 15 mins, and the resulting supernatant was further centrifuged at 8,000g for 20 mins. The 8,000g-supernatant was supplied with Triton X-100 to a final concentration of 0.5 %, and was used as the rat liver extract. All these procedures were carried out at 4 ℃.
The rat liver extract (530μl) was precleaned by centrifugation (20,000g for 15 mins) and incubated with the p47(1-170)-immobilized beads for 2 hrs at 4 ℃. The binding proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The protein bands were excised from the gel and subjected to in-gel digestion with trypsin, followed by mass spectrometry analysis using an LCQ-Deca IT mass spectrometer (Finnigan).