After CRH stimulation, purified splenic B cells were suspended in NP-40 cell lysis buffer (150 mM sodium chloride, 50 mM Tris pH 8.0, 1% NP-40) and protein extraction was performed in the presence of a complete protease and phosphatase inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany). Protein concentrations were determined using a Coomassie protein assay (Bio-Rad, Ivry sur Seine, France).
Complete protease and phosphatase inhibitor mixture
Manufactured by Roche
Sourced in United States, Switzerland
Complete protease and phosphatase inhibitor mixture is a laboratory product designed to prevent the degradation of proteins during extraction and purification processes. It contains a combination of protease and phosphatase inhibitors that work to inactivate a wide range of enzymatic activities, ensuring the integrity of sample proteins.
Automatically generated - may contain errors
Lab products found in correlation
C18 silica cartridge, by Nest Group (3 mentions)
Coomassie protein assay, by Bio-Rad (1 mentions)
Torin1, by Bio-Techne (1 mentions)
Mk2206, by Merck Group (1 mentions)
Scramble sirna, by Santa Cruz Biotechnology (1 mentions)
G6pase α, by Santa Cruz Biotechnology (1 mentions)
Phospho 4e bp1 thr37 46, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
4 protocols using complete protease and phosphatase inhibitor mixture
1
Splenic B Cell Protein Extraction
2
Investigating mTOR Signaling Pathway
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Reagents were obtained from the following sources: torin1 from Tocris Biosciences (Bristol, UK); rapamycin from Calbiochem; MK-2206 from Sigma (St. Louis, MO, USA); SB-415286 from Santa Cruz Biotechnology Inc. (Dallas, TX, USA); Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin, penicillin/streptomycin and Lipofectamine 2000 transfection reagent from Life Technologies (Carlsbad, CA, USA); the complete protease and phosphatase inhibitor mixture from Roche Applied Science (Mannheim, Germany); the antibodies to pan-Akt, phospho-Ser-473 Akt, phospho-Thr-389 S6K1 and phospho-Thr-37/46 4E-BP1 from Cell Signaling Technologies (Danvers, MA, USA); the antibodies to total 4E-BP1, total S6K1 and PEPCK from Santa Cruz Biotechnology Inc.; the PKM1/2 antibody from Thermo (Waltham, MA, USA); and the β-actin antibody from Sigma. siRNA against Raptor, Rictor, PCK1, G6Paseα and scramble siRNA were obtained from Santa Cruz Biotechnology, Inc. All other reagents were from Sigma.
3
Cell Lysis and Protein Extraction
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Cells were treated with lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100) supplemented with complete protease and phosphatase inhibitor mixture (Roche, Basel, Switzerland) and the lysates thus obtained were subjected to SDS-polyacrylamide gel electrophoresis.
4
Protein Extraction and Digestion Protocol
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Cell pellets were resuspended in lysis buffer (8 m urea in 25 mm ammonium bicarbonate) supplemented with complete protease and phosphatase inhibitor mixture (Roche Applied Science). The cells were lysed within a bead beater with the help of 0.1-mm zirconium beads. The cells were disrupted in a Mini bead-beater for 10–12 cycles (45-s pulse with 60-s incubation). Lysates were clarified by centrifugation, and the concentrations were determined by the Bradford assay (Bio-Rad).
50 μg of the sample was first reduced with 5 mm tris(2-carboxyethyl)phosphine and further alkylated with 50 mm iodoacetamide. The samples were diluted to 1 m final urea concentration with 25 mm ammonium bicarbonate buffer and digested with trypsin (1:50, trypsin/lysate ratio) for 16 h at 37 °C. Digests were cleaned using a C18 silica cartridge (The Nest Group, Southborough, MA) according to the manufacturer's protocol and dried using a speed vac. The dried pellet was resuspended in buffer A (5% acetonitrile, 0.1% formic acid).
50 μg of the sample was first reduced with 5 m
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