Protein g a plus agarose beads

Cells were lysed in 1 ml of the whole-cell extract buffer A (50 mM pH7.6 Tris-HCl, 150 mM NaCl, 1%NP40, 0.1 mMEDTA, 1.0 mM DTT, 0.2 mMPMSF, 0.1 mM Pepstatine, 0.1 mM Leupeptine, 0.1 mM Aproine). Five-hundred-microliter cell lysates was used in immunoprecipitation with antibody. In brief, protein was pre-cleared with 30 μl protein G/A-plus agarose beads (Santa Cruz, Biotechnology, Inc. CA) for 1 hour at 4 °C and the supernatant was obtained after centrifugation (5,000 rpm) at 4 °C. Precleared homogenates (supernatant) were incubated with 2 μg of antibody and/or normal mouse/rabbit IgG by rotation for 4 hours at 4 °C, and then the immunoprecipitates were incubated with 30 μl protein G/A-plus agarose beads by rotation overnight at 4 °C, and then centrifuged at 5000 rpm for 5 min at 4 °C. The precipitates were washed five times ×10 min with beads wash solution (50 mM pH7.6 TrisCl, 150 mMNaCl, 0.1%NP-40, 1 mM EDTA) and then resuspended in 60 μl 2 × SDS-PAGE sample loading buffer to incubate for 5–10 min at 100 °C. Then Western blot was performed with a another related antibody indicated in Western blotting.

Cells were lysed in 100 mM KCl, 5 mM MgCl₂, 10 mM HEPES [pH 7.0], 0.5% NP40, 1 mM DTT, 100 units/ml RNase OUT (Invitrogen), 400 μM vanadyl-ribonucleoside complex and protease inhibitors (Roche), clarified and stored on at −80°C. Ribonucleoprotein particle-enriched lysates were incubated with protein G/A-plus agarose beads (Santa Cruz, Biotechnology,Inc.CA) together with primary antibody or normal rabbit IgG for 4 hours at 4°C. Beads were subsequently washed four times with 50 mM TRIS/HCl, pH 7.0, 150 mM NaCl, 1 mM MgCl₂, and 0.05% NP-40, and twice after addition of 1 M Urea. IPs were digested with proteinase K (55°C; 30′) and mRNAs were isolated and then RT-PCR or qRT-PCR was performed according to the manufacturer's instructions.

Protein was pre-cleared with protein G/A-plus agarose beads (Santa Cruz Biotechnology, CA) for 1 hr at 4°C, and the supernatant was obtained after centrifugation (3,000 rpm) at 4°C. Pre-cleared homogenates (supernatant) were incubated with antibodies and/or normal mouse/rabbit immunoglobulin G (IgG) with rotation for 4 hr at 4°C. The immunoprecipitates were incubated with protein G/A-plus agarose beads by rotation overnight at 4°C and then centrifuged at 3,000 rpm for 5 min at 4°C. The precipitates were washed five times for 10 min with beads wash solution and resuspended in loading buffer. Western blotting was performed with related antibodies.

Cells were lysed in 1 ml of the whole-cell extract buffer A (50 mM pH7.6 Tris-HCl, 150 mM NaCl, 1% NP40, 0.1 mM EDTA, 1.0 mM DTT, 0.2 mM PMSF, 0.1 mM Pepstatine, 0.1 mM Leupeptine, 0.1 mM Aproine). Five-hundred-microliter cell lysates was used in immunoprecipitation with antibody. In brief, protein was pre-cleared with 30 μl protein G/A-plus agarose beads (Santa Cruz, Biotechnology, Inc.CA) for 1 hour at 4 °C and the supernatant was obtained after centrifugation (5,000 rpm) at 4 °C. Precleared homogenates (supernatant) were incubated with 2 μg of antibody and/or normal mouse/rabbit IgG by rotation for 4 hours at 4 °C, and then the immunoprecipitates were incubated with 30 μl protein G/A-plus agarose beads by rotation overnight at 4 °C, and then centrifuged at 5000 rpm for 5 min at 4 °C. The precipitates were washed five times ×10 min with beads wash solution (50 mM pH 7.6 Tris Cl, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA) and then resuspended in 60 μl 2 × SDS-PAGE sample loading buffer to incubate for 5–10 min at 100 °C. Then Western blot was performed with a another related antibody indicated in Western blotting.

Cells were lysed in RIRP lysis buffer containing protease inhibitor cocktails (Roch, Diagnostics, Indianapolis IN, USA). Five-hundred-microliter cell lysates was used in immunoprecipitation with antibody. In brief, protein was pre-cleared with 30μl protein G/A-plus agarose beads (Santa Cruz, Biotechnology, Inc. CA) for 1 hour at 4°C and the supernatant was obtained after centrifugation (5, 000rpm) at 4°C. Precleared homogenates (supernatant) were incubated with 2 μg of antibody and/or normal mouse/rabbit IgG by rotation for 4 hours at 4°C, and then the immunoprecipitates were incubated with 30μl protein G/A-plus agarose beads by rotation overnight at 4°C, and then centrifuged at 5000rpm for 5 min at 4°C. The precipitates were washed five times×10min with beads wash solution (50 mM pH7.6 TrisCl, 150mMNaCl, 0.1%NP-40, 1mM EDTA) and then resuspended in 40μl 2×SDS-PAGE sample loading buffer to incubate for 10 min at 100°C. Then Western blot was performed with a another related antibodies.

Cells were lysed in 1 ml of the whole-cell extract buffer A (50mM pH7.6 Tris-HCl, 150mMNaCl, 1%NP40, 0.1mMEDTA,1.0mM DTT,0.2mMPMSF, 0.1mM Pepstatine,0.1mM Leupeptine,0.1mM Aproine). Five-hundred-microliter cell lysates was used in immunoprecipitation with antibody. In brief, protein was pre-cleared with 30μl protein G/A-plus agarose beads (Santa Cruz, Biotechnology, Inc. CA) for 1 hour at 4°C and the supernatant was obtained after centrifugation (5,000rpm) at 4°C. Precleared homogenates (supernatant) were incubated with 2 μg of antibody and/or normal mouse/rabbit IgG by rotation for 4 hours at 4°C, and then the immunoprecipitates were incubated with 30μl protein G/A-plus agarose beads by rotation overnight at 4°C, and then centrifuged at 5000rpm for 5 min at 4°C. The precipitates were washed five times×10min with beads wash solution (50 mM pH7.6 TrisCl,150mMNaCl,0.1%NP-40,1mM EDTA) and then resuspended in 60μl 2×SDS-PAGE sample loading buffer to incubate for 10 min at 100°C. Then Western blot was performed with a another related antibody indicated in Western blotting.