Ripa buffer

Manufactured by Boston BioProducts

Sourced in United States, Switzerland

RIPA buffer is a commonly used cell lysis and protein extraction solution. It is a mixture of ionic and non-ionic detergents that effectively solubilize cellular proteins. The buffer is designed to extract proteins from various sample types, including cells, tissues, and other biological samples.

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Lab products found in correlation

Protease inhibitor cocktail, by Merck Group (28 mentions) Pvdf membrane, by Merck Group (20 mentions) Pvdf membrane, by Bio-Rad (17 mentions) Phosphatase inhibitor cocktail, by Merck Group (16 mentions) Ecl western blotting substrate, by Cytiva (15 mentions) β actin, by Cell Signaling Technology (14 mentions) Nitrocellulose membrane, by Bio-Rad (13 mentions) Protease inhibitor, by Roche (13 mentions) Protease inhibitor cocktail, by Roche (12 mentions) Protease and phosphatase inhibitor, by Roche (11 mentions) β actin, by Merck Group (10 mentions) Protease and phosphatase inhibitor, by Thermo Fisher Scientific (10 mentions) Complete protease inhibitor cocktail, by Roche (9 mentions) Chemidoc mp imaging system, by Bio-Rad (8 mentions) Bca assay, by Thermo Fisher Scientific (8 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (7 mentions) Bca protein assay, by Thermo Fisher Scientific (7 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (7 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (7 mentions) Polyvinylidene difluoride membrane, by Merck Group (7 mentions)

Spelling variants of this product

RIPA lysis buffer (48 citations) Radioimmunoprecipitation assay (RIPA) buffer (4 citations) Phosphate-Buffered RIPA lysis buffer (2 citations)

277 protocols using ripa buffer

Western Blot Analysis of Apoptosis Regulators

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Whole-cell lysates were prepared by lysing cells on ice with 1× RIPA buffer (Boston BioProducts): 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate and water (rest) supplemented with protease inhibitors (1 mM AEBSF, 0.8 μM aprotinin, 0.05 mM bestatin, 0.015 mM E-64, 0.02 mM leupeptin, 0.01 mM pepstatin A). Protein lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane in a wet transfer system for 1 h at 100 V. Membranes were incubated with anti-MCL1 Abs (D2W9E and D35A5, Cell Signaling), anti-BAX Abs (D2E11, Cell Signaling), anti-P18 Abs, DCS-118 from Cell Signaling, anti-β-actin Abs, PA1–21167, from Pierce (Rockford, IL, USA), and anti-CDK6 Ab, (C-21): sc-177 from Santa Cruz at a dilution of 1:1000, at 4 °C overnight. HRP-conjugated secondary Abs were used for detection using ECL2 reagent (Pierce). Immunoblots were visualized on Bio-Rad ChemiDoc MP imaging system.

Whitaker R.H, & Placzek W.J. (2020). MCL1 binding to the reverse BH3 motif of P18INK4C couples cell survival to cell proliferation. Cell Death & Disease, 11(2), 156.

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Protein Extraction and Western Blot

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Cellular protein was extracted by treating 1e7 cells / mL in ice-cold 1 × RIPA buffer (Boston BioProducts), containing protease inhibitors (Roche, Sigma) for 30 min on ice, pelleting cell debris for 15 min at 12,000 × g at 4 °C, then the protein-containing supernatant was collected, and concentrations were measured by BCA assay (Abcam). Proteins were denatured in Laemmli loading buffer at 95 °C for 5 min, and 20 μg / sample were loaded into 4 % - 20 % SDS-PAGE gel for electrophoresis at 80 V for 20 min then 120 V for 70 min. Proteins were transferred to PVDF membranes using a wet transfer at 320 mA for 70 min on ice. Membranes were blocked at room temperature for 1 h using 3 % BSA in TBST buffer, transferred membranes were incubated in 1:1000 diluted primary antibody in TBST buffer with 3 % BSA at 4 °C overnight, then incubated with 1:5000 horseradish peroxidase (HRP)-conjugated secondary antibodies in TBST buffer for 1 hr at room-temperature. The chemiluminescent substrate (Bio-Rad) was added to membranes and immunoblots were imaged using an Amersham Imager 600 (GE Healthcare).

Renauer P., Park J.J., Bai M., Acosta A., Lee W.H., Lin G.H., Zhang Y., Dai X., Wang G., Errami Y., Wu T., Clark P., Ye L., Yang Q, & Chen S. (2023). Immunogenetic metabolomics revealed key enzymes that modulate CAR-T metabolism and function. bioRxiv.

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Western Blot Analysis of Cell Lysates

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Whole cell lysates and nuclear fraction were prepared using RIPA buffer (Boston BioProducts, Ashland, MA) and Nuclear Extract Kit (Active Motif, Carlsbad, CA), respectively in the presence of protease inhibitor cocktail (Millipore Sigma, St. Louis, MO) and Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations of the cell lysates were quantified using a BCA protein assay (Thermo Fisher Scientific) to ensure equal amounts of total protein were loaded in each well of NuPAGE 4–12% Bis-Tris Gel (Thermo Fisher Scientific). Proteins were transferred onto a nitrocellulose membrane and blotted with the appropriate antibodies, followed by the HRP-conjugated secondary antibodies, and the detection was performed using ECL Plus Western blotting detection reagents (GE Healthcare, Chicago, IL). ECL signal was detected using the Azure 300 (Azure Biosystem, Dublin, CA). The antibodies used for WB are listed in key resources table.

Sui H., Chen Q., Yang J., Srirattanapirom S, & Imamichi T. (2022). Manganese enhances DNA- or RNA-mediated innate immune response by inducing phosphorylation of TANK-binding kinase 1. iScience, 25(11), 105352.

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Western Blotting and Immunoprecipitation Methods

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For western blotting, cells were lysed in RIPA buffer (Boston BioProducts) supplemented with protease (Roche) and phosphatase (Roche) inhibitor. Proteins were separated on NuPAGE 4–12% Bis-Tris gradient gels (Invitrogen), transferred to polyvinylidine difluoride membranes (Immobilon P, Millipore) and the blots were probed with the indicated antibodies. For immunoprecipitation, U2OS, DU145, PC3, 293T, and MEF cells were transfected with the indicated expression vectors by using LIPOFECTAMIN 2000 (Life Technologies). Twenty-four hours after transfection, cells were lysed in RIPA buffer with protease (Roche) and phosphatase (Roche) inhibitor. Of total lysates, 500 mg were precleared for 30 minutes at 4°C and then immunoprecipitated with anti-Myc (Cell Signaling Technology 9B11, 1:500), or anti-PTEN (Cell Signaling Technology 9559, 1:500) antibody overnight at 4°C. The Protein-A or Protein-G sepharose beads (GE Healthcare) were then added and incubated for another 2 hours. The immunoprecipitates were washed with RIPA buffer three times. In denaturing conditions, standard Laemmli-Buffer with 5% final concentration of β-mercaptoethanol was added to the samples, which were then boiled and separated on NuPAGE 4–12% Bis-Tris gradient gels (Invitrogen).

Lee Y.R., Chen M., Lee J.D., Zhang J., Lin S.Y., Fu T.M., Chen H., Ishikawa T., Chiang S.Y., Katon J., Zhang Y., Shulga Y.V., Bester A.C., Fung J., Monteleone E., Wan L., Shen C., Hsu C.H., Papa A., Clohessy J.G., Teruya-Feldstein J., Jain S., Wu H., Matesic L., Chen R.H., Wei W, & Pandolfi P.P. (2019). Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway. Science (New York, N.Y.), 364(6441), eaau0159.

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Immunohistochemical and Western Blot Analysis of Biological Samples

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Tissues were fixed in 10% formalin overnight and embedded in paraffin. Immunohistochemical (IHC) and immunofluorescence (IF) staining was performed as previously described^{11 (link),14 (link)}. IHC slides were scanned with Pannoramic Digital Slide Scanner (3DHISTECH) and images were cropped from virtual slides in Pannoramic Viewer. IF slides were imaged with Nikon A1R Confocal Laser Microscope and quantified with ImageJ. Primary antibodies used include CK5 (Covance, PRB-160P), CK8 (Covance, MMS-162P), Ki67 (Fisher, RM-9106-S1), cleaved caspase 3 (Cell Signaling Technology, 9661), Gr-1 (BioLegend, 108401), phospho-S6 (Cell Signaling Technology, 4858). For Western blot analysis, cells or fresh tissues were lysed on ice using RIPA buffer (Boston BioProducts) supplemented with protease and phosphatase inhibitors (Roche). Western blot procedure was performed as previously described^{11 (link),14 (link)}. Primary antibodies used include phospho-Met (Cell Signaling Technology, 3077), phospho-VEGFR2 (Cell Signaling Technology, 3770), phospho-Erk1/2 (Cell Signaling Technology, 4370), phospho-Akt (Cell Signaling Technology, 4060), phospho-mTOR (Cell Signaling Technology, 5536), phospho-p70 S6K (Cell Signaling Technology, 9234), phospho-S6 (Cell Signaling Technology, 4856), and vinculin (Millipore, 05-386).

Lu X., Horner J.W., Paul E., Shang X., Troncoso P., Deng P., Jiang S., Chang Q., Spring D.J., Sharma P., Zebala J.A., Maeda D.Y., Wang Y.A, & DePinho R.A. (2017). Effective Combinatorial Immunotherapy for Castration Resistant Prostate Cancer. Nature, 543(7647), 728-732.

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Western Blot Analysis for ALK Protein

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Total protein was isolated from frozen tissue homogenized in RIPA buffer (Boston BioProducts, Ashland, MA) using OMNI-GLH (OMNI International, Kennesaw, GA); 40 μg of total protein was resolved by SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membranes were incubated overnight at 4˚C with ALK (D5F3, 1:2000, Cell Signaling) or β-actin (1:1000, Cell Signaling) antibodies, followed by anti-rabbit (1:10000, Promega, Madison, WI) HRP-conjugated secondary antibody and signals were detected by ECL (GE Healthcare, Chicago, IL).

Panebianco F., Nikitski A.V., Nikiforova M.N., Kaya C., Yip L., Condello V., Wald A.I., Nikiforov Y.E, & Chiosea S.I. (2019). Characterization of Thyroid Cancer Driven by Known and Novel ALK Fusions. Endocrine-related cancer, 26(11), 803-814.

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Protein Extraction and Western Blot Analysis

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Tissues were digested in RIPA buffer (Boston BioProducts, Ashland, MA) with freshly added protease inhibitors (Sigma) and homogenized at 15,000 rpm for 30 sec. After incubation for 1 h at 4°C, insoluble particles were removed by centrifugation at 14,000 g for 25 min. Protein concentration was quantified using the Bradford assay and then boiled in Laemmli's reducing buffer (Boston BioProducts) for 25 min at 65°C. Protein was electrophoresed on polyacrylamide gels, transferred to a nitrocellulose membrane, and blocked for 2 h in 5% nonfat dry milk (NFDM) in TBS with 0.05% Tween-20 (TBS-T). Primary antibodies of monoclonal anti-rat β-actin at a dilution of 1:2000 (Sigma) and monoclonal anti-rat MHC class I (major histocompatibility complex) antibody of RT1A at a dilution of 1:500 (BD Pharmingen, BD Biosciences, CA) were applied overnight in 1.5% NFDM in TBS-T, followed by horseradish peroxidase–conjugated goat secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature at a dilution of 1:2000. Protein was detected using substrate from SuperSignal West Pico.

Tsuchiya T., Mendez J., Calle E.A., Hatachi G., Doi R., Zhao L., Suematsu T., Nagayasu T, & Niklason L.E. (2016). Ventilation-Based Decellularization System of the Lung. BioResearch Open Access, 5(1), 118-126.

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Cytokine Profiling of Frozen Tumors

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Frozen tumors of each group were prepared using a lysis cocktail buffer (RIPA buffer, Boston Bioproducts Inc., Boston, MA) including phosSTOP, cOmplete (Roche, Indianapolis, IN). Tumors were homogenized with a tissue homogenizer (Qiagen, Valencia, CA), and the suspensions were sonicated using a sonicator (Branson, Notredam, IN). All samples were prepared at a protein concentration of 2μg/ml. The pro-inflammatory Panel 1 Mouse V-PLEX kit was used to measure the concentration of several cytokines – IL-1β, IL-2, IL-6, IL-8, IL-12, IFN-γ, TNF-α and KC-Gro (Meso Scale Discovery, Rockville, MD).

Chen Y., Ramjiawan R.R., Reiberger T., Ng M.R., Hato T., Huang Y., Ochiai H., Kitahara S., Unan E.C., Reddy T.P., Fan C., Huang P., Bardeesy N., Zhu A.X., Jain R.K, & Duda D.G. (2015). CXCR4 inhibition in tumor microenvironment facilitates anti-PD-1 immunotherapy in sorafenib-treated HCC in mice. Hepatology (Baltimore, Md.), 61(5), 1591-1602.

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Protein Extraction and Analysis

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Tissues or cells were lysed in RIPA buffer (Boston BioProducts) supplemented with protease and phosphatase inhibitors (Roche), and processed for Western blot analyses according to standard methodology. Flow cytometry was performed by standard procedures. Fluorescence was recorded using Gallios Flow Cytometer (Beckman Coulter) and analyzed with Kaluza flow cytometry analysis software. See supplementary methods for antibodies used for Western blot and flow cytometry analyses.

Zhang S., Chung W.C., Wu G., Egan S.E., Miele L, & Xu K. (2015). Manic Fringe Promotes A Claudin-Low Breast Cancer Phenotype through Notch-Mediated PIK3CG Induction. Cancer research, 75(10), 1936-1943.

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Hypoxic Regulation of HIF-1α Expression

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MGG123 cells were treated with dimethyl sulfoxide (DMSO), digoxin (1 μM)
or ouabain (1 μM) and placed in an incubator under normoxia, (21%
O₂), or hypoxia, (4%), for 12 hours. Cells were collected and lysed
in RIPA buffer (Boston Bioproducts, Ashland, MA) with protease inhibitors (Complete Mini;
Roche Diagnostics, Mannheim, Germany). Protein concentrations were determined by modified
Bradford assay (Bio-Rad, Hercules, CA). Proteins (20 μg) were separated on a
SDS-polyacrylamide gel, transferred to polyvinylidene difluoride membranes (Bio-Rad), and
incubated with primary antibodies, anti-HIF-1α (BD Biosciences) or anti-Actin
(Sigma Aldrich), at 4°C overnight. Membranes were washed and incubated with
respective secondary antibodies (1:5000, anti-mouse horseradish peroxidase
[HRP] or anti-rabbit HRP, Promega, Madison, WI) at room temperature for 1
hour. The proteins were visualized using the ECL plus Western blotting detection system
(GE Healthcare, Little Chalfont, UK), on Kodak films.

Nigim F., Cavanaugh J., Patel A.P., Curry WT J.r., Esaki S.I., Kasper E.M., Chi A.S., Louis D.N., Martuza R.L., Rabkin S.D, & Wakimoto H. (2015). Targeting Hypoxia-inducible Factor 1α in a New Orthotopic Model of Glioblastoma Recapitulating the Hypoxic Tumor Microenvironment. Journal of neuropathology and experimental neurology, 74(7), 710-722.

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