Anti ddddk tag mab magnetic agarose m185 10

The cells of the stable Lamtor1-3×FLAG clone were harvested and lysed with buffer previously described (13 (link)). Briefly, the lysis buffer consisted of 25 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100. After 30 min of incubation on ice, the cells were mechanically broken and subsequently centrifuged at 16,000g for 10 min at 4 °C. The supernatant was immunoprecipitated at 4 °C, 30 min on a rotary shaker, using anti-DDDDK-tag mAb-magnetic agarose (M185-10, Medical & Biological Laboratories) for immunoprecipitation. After washing four times with Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.5), elution was performed with 8 M urea buffer by shaking at 37 °C for 10 min. For Western blotting, a 1:1 mixture of the eluted sample and 2× Laemmli sample buffer (Bio-Rad Laboratories) containing 2-mercaptoethanol was prepared, and the mixture was boiled for 5 min at 95 °C.