Hrp conjugated antibodies

Mouse ESCs were lysed with RIPA buffer (1% NP-40, 0.1% SDS, 0.5% Deoxycholate in PBS pH 7.0) containing 1x complete protease inhibitors (Roche). Protein was quantified using the Pierce BCA protein assay (Thermo Scientific), resolved on Novex gradient denaturing PAGE gels (Life Technologies) and transferred in 20% methanol to PVDF membrane (Millipore). Membranes were blocked with 5% skim milk in TBS-T and incubated with primary antibodies overnight. Blots were washed in TBS-T. Secondary HRP-conjugated antibodies were purchased from GE Healthcare.

BMDM and BMDC cultures were washed with ice-cold DPBS and lysed on ice in a buffer containing 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.27 M sucrose, 0.1% β-mercaptoethanol, and protease and phosphatase inhibitors (Roche). Tissues were snap frozen in liquid nitrogen prior to lysis. For immunoblots, equal amounts of protein (10 μg) in LDS sample buffer (Invitrogen) were subjected to electrophoresis on 4–12% Bis-Tris polyacrylamide gels (Novex, Life Technologies) and transferred to PVDF membranes (Millipore). For immunopreciptations, cell lysates were incubated overnight (ON) at 4°C with indicated antibodies. Protein G-Sepharose (Invitrogen) was added for 2 h and immunoprecipitates (IPs) were washed twice with 1 ml of lysis buffer containing 0.5 M NaCl and once with 1 ml of 50 mM Tris-HCl pH 7.5. IPs were eluted in LDS buffer, separated on polyacrylamide gels and transferred to PVDF membranes. Membranes were blocked in TBST (50 mM Tris-HCl pH 7.5, 0.15 M NaCl, and 0.25% Tween-20) containing 5% BSA and immunoblotted ON at 4°C with primary antibodies diluted 1000-fold in blocking buffer. Blots were then washed in TBST and incubated for 1 h at 22–24°C with secondary HRP-conjugated antibodies (GE Healthcare) diluted 5000-fold in 5% skim milk in TBST. After repeating the washes, signal was detected with enhanced chemiluminescence reagent.

For Western blot analysis, cells were lysed in cold FLAG lysis buffer [50 mM Tris-HCl (pH 7.3), 137 mM NaCl, 10 mM NaF, 0.5 mM EDTA, 1% Triton X-100, 0.2% Sarkosyl, 10% glycerol, and freshly supplemented protease inhibitor cocktail (Sigma)]. Protein concentration was determined by the Bradford method using Bio-Rad protein assay before proteins were equally loaded and separated in polyacrylamide gels. Nitrocellulose membranes were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline with Tween-20 [20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.1% Tween-20], incubated with indicated primary antibodies, then secondary HRP-conjugated antibodies (GE Healthcare) (1:3000; diluted in 2% (w/v) nonfat dry milk in Tris-buffered saline with 0.1% Tween-20), and detected on autoradiographic films after incubating with ECL (GE Healthcare) or SuperSignal West Dura reagents (Thermo scientific).