Goat anti igm hrp

HBL1, TMD8, OCI-Ly10 and OCI-Ly19 cells were lysed at 10⁷ cells per ml in a modified RIPA buffer (0.5% Triton X-100, 0.25% deoxycholate, 0.025% SDS, 10 mM Tris, pH 8.0, 100 mM NaCl, 10 mM EDTA, 1 mM Na₃VO₄, 30 mM pyrophosphate, 10 mM glycerophosphate, 1 mM AEBSF, 0.02 U ml⁻¹ aprotinin and 0.01% NaN₃) for 10 min. on ice. Lysates were cleared by centrifugation at 14,000xg for 20 min. at 4°C. IgM was immunoprecipitated by incubating lysates on ice for 1 hour with 10 μg of biotin-labeled goat anti-human IgM (Jackson Immunoresearch), followed by the addition of 35 μl of pre-washed streptavidin-agarose beads (Invitrogen) and rotated for 30 min. at 4°C. Beads were washed 3X with cold 1X RIPA buffer, then solubilized by adding 2X LDS sample buffer (Invitrogen) with 1% β-mercaptoethanol and boiled for 5 min. Samples were separated on a 10% polyacrylamide gel and transferred to Immobilon-p PVDF membrane (Millipore) for western blot analysis. Membranes were probed with rabbit anti-TLR9 monoclonal XP, rabbit anti-TLR7 (Cell Signaling Technologies), rabbit anti-TLR4 (Santa Cruz Biotechnology) and goat anti-IgM-HRP (Bethyl).