Radioimmunoprecipitation assay buffer

Manufactured by Boston BioProducts

Sourced in United States

Radioimmunoprecipitation assay (RIPA) buffer is a commonly used detergent-based cell lysis and extraction buffer. It is designed to solubilize proteins and maintain their native conformation for further analysis.

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Lab products found in correlation

Oxyblot, by Merck Group (2 mentions) Oxyblot protein oxidation detection kit, by Merck Group (2 mentions) Neutralization solution, by Merck Group (2 mentions) Protease inhibitor, by Merck Group (2 mentions) Phosphatase inhibitor, by Merck Group (2 mentions) C digit blot scanner, by LI COR (2 mentions) Ecl western blotting substrate, by Cytiva (2 mentions) β actin, by Abcam (2 mentions) Polyvinylidene difluoride membrane, by Merck Group (2 mentions) Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions) Glucose assay kit, by Merck Group (1 mentions) Erk1 2, by Cell Signaling Technology (1 mentions) Protein assay dye reagent, by Bio-Rad (1 mentions) Acrylamide gel, by Bio-Rad (1 mentions) Polyvinylidene fluoride membrane, by Merck Group (1 mentions) Collagen 1, by Abcam (1 mentions) β actin, by Merck Group (1 mentions) N cadherin, by Santa Cruz Biotechnology (1 mentions) Vegfr2, by Cell Signaling Technology (1 mentions) Image lab software, by Bio-Rad (1 mentions)

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Radioimmunoprecipitation assay (RIPA) buffer (4 citations)

24 protocols using radioimmunoprecipitation assay buffer

Abscess Tissue Glucose Quantification

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Abscess tissues were carefully dissected away from healthy tissues and were homogenized in 500 μl of radioimmunoprecipitation assay buffer (Boston BioProducts). Abscess homogenates were subsequently centrifuged twice at 12,000g for 15 min, and protein content from the clarified supernatants was quantified using a bicinchoninic acid assay (Thermo Fisher Scientific). Glucose from clarified supernatants containing equivalent amounts of protein was quantified using the glucose assay kit from EMD Millipore (Billerica, MA) following the manufacturer instructions.

Thurlow L.R., Stephens A.C., Hurley K.E, & Richardson A.R. (2020). Lack of nutritional immunity in diabetic skin infections promotes Staphylococcus aureus virulence. Science Advances, 6(46), eabc5569.

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Oxyblot Analysis of Ischemic Myocardium

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Oxyblot (Millipore) was performed on the ischemic and nonischemic myocardial tissue as per manufacturer protocol. Briefly, tissue was lysed in radioimmunoprecipitation assay buffer (Boston BioProducts) and 2 aliquots of each specimen were analyzed (1 aliquot was subjected to a derivatization reaction, while the other aliquot served as a negative control). A 5‐μL protein sample derived from each experimental animal was divided into 2 eppendorf tubes and then denatured with 12% sodium dodecyl sulfate. For the positive control, 2,4‐dinitrophenylhydrazine solution was used to derivatize the samples. Derivatization control solution was used in the control samples. Both sets of samples were incubated at room temperature for 15 minutes after which neutralization solution was added. Finally, the 2 samples were loaded onto a polyacrylamide gel and followed by SDS‐PAGE, Western blot transfer, and immunodetection.

Aboulgheit A., Potz B.A., Scrimgeour L.A., Karbasiafshar C., Shi G., Zhang Z., Machan J.T., Schorl C., Brodsky A.S., Braga K., Pfeiffer M., Gao M., Cummings O., Sodha N.R., Abid M.R, & Sellke F.W. (2021). Effects of High Fat Versus Normal Diet on Extracellular Vesicle–Induced Angiogenesis in a Swine Model of Chronic Myocardial Ischemia. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 10(4), e017437.

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Western Blot Analysis of Vascular Proteins

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Cells were lysed in radioimmunoprecipitation assay buffer (Boston BioProducts). Proteins were titrated using Bio-Rad Protein Assay Dye Reagent (Bio-Rad). 20 ng of proteins was loaded on a 4–12% acrylamide gel (Bio-Rad) and then transferred on a polyvinylidene fluoride membrane (Millipore). Primary antibodies used were Collagen-I, sm22 (Abcam), N-cadherin (Santa Cruz Biotechnology), VEGFR2, eNOS, ERK1/2 (Cell Signaling), and β-actin (Sigma-Aldrich).

Ricard N., Scott R.P., Booth C.J., Velazquez H., Cilfone N.A., Baylon J.L., Gulcher J.R., Quaggin S.E., Chittenden T.W, & Simons M. (2019). Endothelial ERK1/2 signaling maintains integrity of the quiescent endothelium. The Journal of Experimental Medicine, 216(8), 1874-1890.

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Quantitative Western Blotting Analysis

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For Western blotting, whole‐cell lysates were prepared using radioimmunoprecipitation assay buffer (Boston Bio Products, Ashland, MA) with Halt proteinase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA). Protein concentration was measured using a BCA Protein Assay kit (Pierce, Rockford, MA) and a Nanodrop 8000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Equal amounts (30 μg) of proteins were separated onto 4% to 10% mini‐PROTEAN TGX gels (Biorad, Hercules, CA) under reducing and denaturing conditions and transferred to polyvinylidene difluoride membranes. The membranes were blotted with a 1:200 dilution of HuR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. An anti‐GAPDH (1:5000 dilution; Thermo Fisher Scientific, Waltham, MA) or an anti‐tubulin (Sigma) antibody was used as a loading control for all studies. Goat anti‐rabbit or anti‐mouse horseradish peroxidase antibodies (Santa Cruz Biotechnology) were used at a dilution of 1:5000. Proteins were visualized using Clarity Western ECL Blotting Substrate (Biorad). Band intensities were detected by ChemiDoc MP Imaging System and analyzed by Image Lab software (Biorad).

Zhou A., Shi G., Kang G., Xie A., Liu H., Jiang N., Liu M., Jeong E, & Dudley SC J.r. (2018). RNA Binding Protein, HuR, Regulates SCN5A Expression Through Stabilizing MEF2C transcription factor mRNA. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 7(9), e007802.

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Comprehensive Antibody and Reagent List

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Specific reagents used in this study were actinomycin D, G418, hygromycin B, puromycin, sodium butyrate, TriPure Isolation Reagent solution, and valproic acid from Millipore Sigma, doxycycline and NuPAGE^™ LDS Sample Buffer (4×) from Thermo Fisher Scientific, radioimmunoprecipitation assay buffer (RIPA) from Boston Bioproducts (Ashland, MA), and LipoD293^™ In Vitro DNA Transfection Reagent from SignaGen Laboratories (Frederick, MD). Following antibodies were used in this report: affinity-purified rabbit polyclonal anti-ORF57 N-terminal (in house), mouse monoclonal anti-ORF57 N-terminal, and affinity-purified rabbit polyclonal anti-ORF57 C-terminal antibodies were from Rockland Immunochemicals. Rabbit polyclonal anti-AKT, rabbit monoclonal anti-p-AKT, rabbit monoclonal anti-c-FOS (9F6), mouse monoclonal anti-GAPDH, and rabbit polyclonal anti-c-JUN antibodies were purchased from Cell Signaling Technology. Rabbit polyclonal anti-RTA was a gift of Dr. Yoshi Izumiya (UC Davis). Mouse monoclonal anti-myc (9E10) was a gift of Dr. Xuefeng Liu (the Ohio State University). Mouse polyclonal anti-AEN was from Millipore Sigma, rabbit polyclonal anti-RGS2 from Abcam, rabbit polyclonal anti-ZFC3H1(MTR4) antibody from Novus biologicals, and rabbit IgG isotype, mouse IgG isotype were from Thermo Fisher Scientific.

Majerciak V., Alvarado-Hernandez B., Ma Y., Duduskar S., Lobanov A., Cam M, & Zheng Z.M. (2024). KSHV promotes oncogenic FOS to inhibit nuclease AEN and transactivate RGS2 for AKT phosphorylation. bioRxiv.

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Western Blot Protein Expression Analysis

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Cells were lysed in radioimmunoprecipitation assay buffer (Boston
Bioproducts) with protease inhibitors (Roche). Lysates were resolved on
4–12% Bis-Tris SDS-PAGE gels, and transferred to polyvinylidene
difluoride membranes. Membranes were blocked with 5% nonfat milk in
Tris-buffered saline with 0.05% Tween-20 (TBS-T) for 1h, incubated with primary
antibody (1:1000; or 1:4000 β-actin) overnight at 4°C or 1h at RT
(β-actin), washed three times with TBS-T, incubated with HRP-conjugated
secondary antibody (1:2000) for 1h at RT, washed three times with TBS-T,
developed using enhanced chemiluminescence reagents, and imaged with the Bio-Rad
ChemiDoc-XRS imager. Densitometry was performed with Quantity One software
(Bio-Rad).

Ramanujan S.A., Cravens E.N., Krishfield S.M., Kyttaris V.C, & Moulton V.R. (2021). Estrogen‐Induced hsa‐miR‐10b‐5p Is Elevated in T Cells From Patients With Systemic Lupus Erythematosus and Down‐Regulates Serine/Arginine‐Rich Splicing Factor 1. Arthritis & Rheumatology (Hoboken, N.j.), 73(11), 2052-2058.

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Evaluating BGJ-398 Efficacy in Xenograft Mice

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Six- to eight-week-old BALB/c nu⁻/nu⁻ mice (Cox Laboratories, Massachusetts General Hospital) were maintained in accordance with the institutional guidelines of the Massachusetts General Hospital animal care facility. HuH-7 (S2 signature) and SK-Hep (non-S2 signature) were trypsinized and suspended in a 1:1 solution of 4°C PBS:Matrigel Matrix Solution (BD Biosciences) at a concentration of 5 × 10⁶ living cells/100uL solution. Mice were anesthetized with ketamine/xylazine and 100uL of cell suspension was injected subcutaneously into the right flank. When tumors reached 100 mm³, mice were randomized to receive BGJ-398 (30mg/kg) in 100 μL of 0.5% methylcellulose daily by oral gavage, or 100 uL of 0.5% methylcellulose alone. Tumor volumes were measured every other day for 14 days using a calipers and the mathematical approximation of an ellipsoid: V = 0.52 × length × width × height. Mice were euthanized at the end of the study. Tumors were immediately bisected with half the tissue fixed in 10% formalin and half snap frozen in liquid nitrogen and stored at −80C. Frozen portions of tumors were thawed, minced and sonicated in radioimmunoprecipitation assay buffer (Boston BioProducts), before proceeding with western blotting as above.

Schmidt B., Wei L., DePeralta D.K., Hoshida Y., Tan P.S., Sun X., Sventek J.P., Lanuti M., Tanabe K.K, & Fuchs B.C. (2015). Molecular Subclasses of Hepatocellular Carcinoma Predict Sensitivity to Fibroblast Growth Factor Receptor Inhibition. International journal of cancer, 138(6), 1494-1505.

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Quantifying Oxidative Stress in Ischemic Myocardium

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Oxidative stress in ischemic myocardial tissue was determined using an Oxyblot Protein Oxidation Detection Kit (MilliporeSigma, Burlington, MA), which detects carbonyl groups introduced into proteins by oxidative reactions. Tissue was lysed in radioimmunoprecipitation assay buffer (Boston Bioproducts, Milford, MA). A sample of 20 μg of protein was denatured with 12% sodium dodecyl sulfate, subjected to 2,4‐dinitrophenylhydrazine solution to derivatize the samples, incubated at room temperature for 15 minutes, and neutralized with neutralization solution as per the manufacturer's protocol (MilliporeSigma, Burlington, MA). The samples were fractionated on a 4% to 12% Bis‐Tris gel, transferred to a nitrocellulose membrane, incubated with primary and secondary antibodies provided in the kit, processed for chemiluminescent detection, and captured with a digital camera system (Bio‐Rad ChemiDoc MP, Life Science, Hercules, CA). Densitometric analysis of total column band intensity was performed using National Institutes of Health ImageJ software.

Sabe S.A., Xu C.M., Sabra M., Harris D.D., Malhotra A., Aboulgheit A., Stanley M., Abid M.R, & Sellke F.W. (2022). Canagliflozin Improves Myocardial Perfusion, Fibrosis, and Function in a Swine Model of Chronic Myocardial Ischemia. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 12(1), e028623.

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Western Blot Analysis Protocol

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Western blot analysis was performed according to the protocol of our previous study (Son et al., 2020 (link)). The samples for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) were prepared by extracting proteins from 3T3‐L1 cells with radio‐immunoprecipitation assay buffer (Boston Bio Products) containing protease inhibitor (Sigma‐Aldrich) and phosphatase inhibitor (Sigma‐Aldrich) and quantifying the protein amounts by BCA assay (Thermo Fisher Scientific). After sample preparation, the protein (25 μg/well) was separated by SDS‐PAGE, and then proteins separated on the gel were transferred to the polyvinylidene fluoride (PVDF) membrane. After the transfer, the PVDF membrane was blocked with 5% nonfat milk in Tris‐buffered saline containing .05% Tween 20 (TBS‐T) at room temperature for 1 hr, and then, the PVDF membranes were treated with the primary antibodies in 5% bovine serum albumin in TBS‐T at 4ºC for overnight. After the primary antibody treatment was completed, the PVDF membranes were treated with the secondary antibodies in 5% nonfat milk in TBS‐T at room temperature for 1 hr. Chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences) and visualized using LI‐COR C‐DiGit Blot Scanner (Li‐COR Biosciences). The density of Western blot bands was calculated using the software UN‐SCAN‐IT gel version 5.1 (Silk Scientific Inc.).

Geum N.G., Son H.J., Yeo J.H., Yu J.H., Choi M.Y., Lee J.W., Baek J.K, & Jeong J.B. (2021). Anti‐obesity activity of Heracleum moellendorffii root extracts in 3T3‐L1 adipocytes. Food Science & Nutrition, 9(11), 5939-5945.

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Quantitative Protein Analysis of Liver Tissue

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Tissue lysates were prepared from frozen liver with radio-immunoprecipitation assay buffer (Boston Bioproducts, Ashland, MA, USA) containing HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA). Equal amounts of protein from each sample (20–30 μg) were separated on 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and subsequently transferred to polyvinylidene fluoride or nitrocellulose membranes. Membranes were then blocked in 5% milk and incubated with anti-osteopontin (R&D Systems, Minneapolis, MN, USA) and anti-GAPDH (loading control; Cell Signaling Technology, Danvers, MA, USA). Species-appropriate horseradish peroxidase-conjugated secondary antibodies (Millipore, Billerica, MA, USA) were used to detect antigen–antibody complexes. Blots were developed using Luminata Classico (Millipore) and exposed to X-ray film. Label-free quantitation was performed using the spectral count approach, in which the relative protein quantitation is measured by comparing the number of tandem mass spectrometry (MS/MS) spectra identified from the same protein in each of the multiple liquid chromatography (LC)/MS/ MS data sets. Scaffold was used for quantitation analysis.

Thompson M.D., Cismowski M.J., Trask A.J., Lallier S.W., Graf A.E., Rogers L.K., Lucchesi P.A, & Brigstock D.R. (2016). Enhanced Steatosis and Fibrosis in Liver of Adult Offspring Exposed to Maternal High-Fat Diet. Gene Expression, 17(1), 47-59.

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