Radio immunoprecipitation buffer

Manufactured by Boston BioProducts

Radio-immunoprecipitation buffer is a solution used in the process of radio-immunoprecipitation assay (RIPA) to isolate and purify target proteins from cell lysates. It facilitates the extraction and solubilization of proteins, while maintaining their native structure and interactions.

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Lab products found in correlation

Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions) Sodium orthovanadate, by Merck Group (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions) Sodium fluoride, by Merck Group (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions) Cleaved parp, by Cell Signaling Technology (1 mentions) Ecl kit, by Cytiva (1 mentions) Nampt, by Fortis Life Sciences (1 mentions) Vinculin, by Thermo Fisher Scientific (1 mentions) Hexokinase 2, by Cell Signaling Technology (1 mentions) Phospho ampk, by Cell Signaling Technology (1 mentions) C myc, by Cell Signaling Technology (1 mentions) Of protease and phosphatase inhibitors, by Roche (1 mentions) N myc, by Cell Signaling Technology (1 mentions) Phospho raptor, by Cell Signaling Technology (1 mentions) Naprt1, by Merck Group (1 mentions) Phospho s6, by Cell Signaling Technology (1 mentions) Phospho mtor, by Cell Signaling Technology (1 mentions) Phospho 4ebp1, by Cell Signaling Technology (1 mentions)

4 protocols using radio immunoprecipitation buffer

Protein Extraction from HLMVEC Cells

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At the end of the experiment, HLMVEC were harvested and lysed with radio-immunoprecipitation buffer (Boston Bioproducts, Ashland, MA), supplemented with protease inhibitor cocktail, 200 mM phenylmethylsulfonylfluoride, 1 mM EDTA, 1 mM sodium-fluoride, and 1 mM sodium-orthovanadate (all from Sigma-Aldrich) as previously described [26 (link),27 (link)].

Piegeler T., Dull R.O., Hu G., Castellon M., Chignalia A.Z., Koshy R.G., Votta-Velis E.G., Borgeat A., Schwartz D.E., Beck-Schimmer B, & Minshall R.D. (2014). Ropivacaine attenuates endotoxin plus hyperinflation-mediated acute lung injury via inhibition of early-onset Src-dependent signaling. BMC Anesthesiology, 14, 57.

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Immunoblotting Assay for Protein Expression

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Cells were lysed in radioimmunoprecipitation buffer (Boston Bioproducts) with a cocktail of protease and phosphatase inhibitors (Roche). 12.5 µg of protein was separated by 4–20% SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. After blocking with 5% nonfat dry milk in TBST (20 mM Tris [pH, 7.5], 150 mM NaCl, 0.1% Tween20), membranes were incubated at 4C overnight with primary antibodies. After washing and incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology), blots were washed, and signals were visualized with an ECL kit (Amersham Bioscience). Primary antibodies: c-Myc, N-Myc, Hexokinase II (HKII), PKM2, LDHA, cleaved-PARP, phospho-AMPK, phospho-Raptor, phospho-mTOR, phospho-S6, phospho-4E-BP1, Cleaved PARP, actin (Cell Signaling); NAMPT (Bethyl Laboratories); Naprt1 (Sigma Aldrich); and Vinculin (Thermo Scientific).

Tateishi K., Iafrate A.J., Ho Q., Curry W.T., Batchelor T.T., Flaherty K.T., Onozato M.L., Lelic N., Sundaram S., Cahill D.P., Chi A.S, & Wakimoto H. (2016). Myc-driven glycolysis is a therapeutic target in glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(17), 4452-4465.

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Western Blot Analysis of MGMT and MSH6

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005 GSCs were pelleted and lysed in radioimmunoprecipitation buffer (Boston Bioproducts) with a cocktail of protease and phosphatase inhibitors (Roche). Protein (20 µg) was separated by 4%–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes by electroblotting. Western blotting was performed with primary antibodies to MGMT (Biovision, Cat. # 3820–100) and MSH6 (Cell Signaling Technology, Cat. # 3995). Primary antibodies to β-actin (Cell Signaling Technology, Cat. # 4970) and Vinculin (Cell Signaling Technology, Cat. # 13901) were used as loading controls.

Saha D., Rabkin S.D, & Martuza R.L. (2020). Temozolomide antagonizes oncolytic immunovirotherapy in glioblastoma. Journal for Immunotherapy of Cancer, 8(1), e000345.

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Cell Lysis and Protein Extraction

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After the indicated incubation times, HLMVECs were washed once with ice-cold phosphate-buffered saline (Gibco Invitrogen), scraped from the bottom of the plate, collected, and lysed with radio-immunoprecipitation buffer (Boston Bioproducts, Ashland, MA) supplemented with protease inhibitor cocktail, 200 mM of phenylmethylsulfonyl fluoride, 1 mM of EDTA, 1 mM of sodium fluoride, and 1 mM of sodium orthovanadate (all from Sigma-Aldrich) as previously described.^{22 (link),23 (link)}

Piegeler T., Votta-Velis E.G., Bakhshi F.R., Mao M., Carnegie G., Bonini M.G., Schwartz D.E., Borgeat A., Beck-Schimmer B, & Minshall R.D. (2014). Endothelial Barrier Protection by Local Anesthetics: Ropivacaine and Lidocaine Block Tumor Necrosis Factor-α–induced Endothelial Cell Src Activation. Anesthesiology, 120(6), 1414-1428.

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