Clean blot ip detection kit

After protein estimation using the Lowry method, indicated concentrations of purified METTL23-His8-tag protein (22.6 kDa) were added to 10 μl of 4× NuPAGE LDS sample buffer contain ing 2 μl sample reducing agent (Invitrogen, Austria) and heated (70°C, 10 min). Purified METTL23-His8-tag protein and immunoprecipitates were separated by electrophoresis on 12% Bis–Tris gel and transferred to nitrocellulose membranes. Membranes were blocked with 5% (w/v) non-fat milk in TBST (Tris-buffered saline containing Tween 20) (25°C, 2 h) and incubated with either (i) rabbit polyclonal anti-METTL23 antiserum (1:2000 in 5%, w/v, BSA), (ii) rabbit polyclonal anti-His-tag (C-terminal) antibody (1:1000 in 5%, w/v, BSA, Relia Tech, Germany) and (iii) rabbit sequence-specific anti-METTL23 (C17orf95) antibody (1:300 in 5%, w/v, BSA, Abgent, Germany, raised against a synthetic peptide; position 137–166 amino acids) (4°C, overnight). Immunoreactive bands were visualized with either Clean-Blot IP Detection Kit [horseradish peroxidase (HRP)] (1:100 000 in 5%, w/v, non-fat milk in TBST, Thermo scientific, Austria) or HRP-conjugated goat anti-rabbit IgG (1:100 000 in 5%, w/v, non-fat milk in TBST) (25°C, 2 h) followed by Super Signal West Pico Chemiluminescent substrate (Thermo Scientific, Austria) and developed by Bio-Rad ChemiDoc MP Imaging System.

Cells were lysed with lysis buffer containing 50 mM HEPES, pH7.4, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF and 1 x protease inhibitor cocktail (Roche). Cell debris and unbroken cells were removed by centrifugation at 10, 000 g for 10 min. Clarified lysates were incubated with antibodies overnight followed by addition of Protein A conjugated agarose beads (Life Technologies). After incubation for additional 1.5 hr with agitation, beads were washed four times with lysis buffer, once with 20 mM Tris-HCl (pH7.4) and boiled for 5 min in 60 μl of 2x SDS sample buffer. Samples were subjected to SDS-PAGE. Western blotting was performed by standard protocols and developed using ECL 2 reagents (Pierce). Clean-blot IP detection kit from Thermo-Fisher Scientific (#21232) was used to detect immunoprecipitated RagC (Fig. 5C). Antibody concentrations were optimized with various dilutions to ensure that the blotting signals are linear to the levels of loaded antigens. Quantitative analysis of the blots was performed with densitometry scanning. Data from at least three independent experiments were analyzed.

MC3T3-E1 cells grown in 150-mm dishes were serum-starved for 12 hours and then stimulated with or without 0.3 µg/ml APN for 30 minutes. Cells were then lysed on ice with lysis buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, 45 mM octyl glucoside) supplemented with protease and phosphatase inhibitors cocktails. IP was performed with Protein A/G Magnetic Beads (EMD Millipore, Billerica, MA) and 1 µg of specified antibodies at 4°C overnight. The immunoprecipitates were washed with lysis buffer and further analyzed by polyacrylamide gel electrophoresis and Western blot analyses. The Clean-Blot IP Detection Kit (Thermo Scientific) was used as the secondary antibody. Mouse IgG (Millipore) was used as a negative control in all IP experiments.