Fig. 1.

Specific interaction of TrkA with EIIA^Ntr. Ligand fishing to search for protein factor(s) interacting with EIIA^Ntr. Crude extract prepared from MG1655 grown in 500 ml of LB to A₆₀₀ of 2.0 was mixed with 2 mg of either purified His–EIIA^Ntr (lane 1) or EIIA^Ntr (lane 2), and these mixtures were passed through metal-affinity chromatography columns containing 500 μl of TALON resin. After washing the columns with 20 mM Hepes buffer (pH 7.5) containing 200 mM NaCl three times (2 ml), proteins bound to the resin were eluted with the same buffer (500 μl) containing 200 mM imidazole, and the eluates were analyzed by SDS/PAGE followed by staining with Coomassie brilliant blue (Bio-Rad, Hercules, CA). In-gel digestion followed by MALDI-TOF analysis revealed that the protein band bound specifically to His–EIIA^Ntr corresponded to TrkA (marked with an arrow). Tefco (Tokyo, Japan) Wide Range protein standards were used as molecular mass markers (lane M).