Ripa lysis buffer

Manufactured by Merck Group

Sourced in United States, Germany, China, Switzerland, Macao, Canada, United Kingdom, France, Sao Tome and Principe, Italy, Japan, India

RIPA lysis buffer is a laboratory reagent used for the extraction and solubilization of proteins from cells and tissues. It is a detergent-based buffer that disrupts cell membranes and aids in the release of cellular proteins. The buffer contains a combination of ionic and non-ionic detergents, as well as other components that help maintain the stability and activity of the extracted proteins.

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Lab products found in correlation

Pvdf membrane, by Merck Group (144 mentions) Bca protein assay kit, by Thermo Fisher Scientific (137 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (121 mentions) Protease inhibitor cocktail, by Roche (78 mentions) Protease inhibitor cocktail, by Merck Group (67 mentions) Bca kit, by Thermo Fisher Scientific (66 mentions) Protease inhibitor, by Merck Group (41 mentions) Protease inhibitor, by Roche (37 mentions) Odyssey infrared imaging system, by LI COR (34 mentions) Pvdf membrane, by Bio-Rad (34 mentions) β actin, by Merck Group (32 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (32 mentions) Image lab software, by Bio-Rad (31 mentions) Nitrocellulose membrane, by Bio-Rad (31 mentions) Bca protein assay kit, by Beyotime (29 mentions) Protease and phosphatase inhibitor, by Merck Group (26 mentions) Bovine serum albumin (bsa), by Merck Group (25 mentions) Gapdh, by Cell Signaling Technology (24 mentions) Bca protein assay, by Thermo Fisher Scientific (24 mentions) Bradford assay, by Bio-Rad (23 mentions)

Spelling variants of this product

RIPA buffer (4162 citations) Lysis buffer (324 citations) RIPA cell lysis buffer (32 citations) RIPA lysis (7 citations) RIPA lysis buffer 10× (7 citations) Modified RIPA lysis buffer (5 citations) RIPA protein lysis buffer (4 citations) RIPA lysis buffer cell lysis buffer (2 citations) RIPA lysis buffer and phosphatase (2 citations) RIPA lysis buffer (R0278), (2 citations)

1 469 protocols using ripa lysis buffer

Evaluating Salmonella Invasion and Replication

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RAW 264.7 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and was grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich; St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé France), and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; Gibco; Waltham, MA, USA) at 37°C in the presence of 5% CO₂. RAW 264.7 cell (3 x 10⁴ cells per well) was seeded on 48-well plates (SPL Life Sciences, Pocheon, Republic of Korea) and incubated for 16 h. Attenuated Salmonella candidates were cultured in LB at OD₆₀₀ of 1.0 and harvested. The strains were treated to RAW 264.7 cells at multiplicity of infection (MOI) of 10 and incubated for 2 h at 37°CC. The cells were washed with PBS (Welgene, Gyeongsan, Republic of Korea) thrice and transferred to DMEM supplemented with 10% FBS and 100 μg/mL of gentamicin to eliminate extracellular bacteria. After 2 h incubation, the cells were washed three times with PBS and treated with RIPA lysis buffer (Sigma) for invasion assay. For replication assay, the cells were incubated with DMEM supplemented with 10% FBS and 10 μg/mL gentamicin to prevent the leakage of intracellular bacteria. After 16 h incubation, cells were treated with RIPA lysis buffer (Sigma). Subsequently, lysis samples were serially diluted with PBS and spotted on the LB agar plate.

Ji H.J., Jang A.Y., Song J.Y., Ahn K.B., Han S.H., Bang S.J., Jung H.K., Hur J, & Seo H.S. (2022). Development of Live Attenuated Salmonella Typhimurium Vaccine Strain Using Radiation Mutation Enhancement Technology (R-MET). Frontiers in Immunology, 13, 931052.

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Protein Extraction from Heart Tissues

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Heart tissues were homogenized in RIPA lysis buffer (Sigma; Cat. No. R0278) including a protease inhibitor cocktail (Roche; Cat. No. 11873580001). H9c2 cells were washed with cold PBS (Aurogene; AU‐L0615), scraped in 150 µL of cold RIPA lysis buffer (Sigma; Cat. No. R0278) including a protease inhibitor cocktail (Roche; Cat. No. 1187358000) and centrifuged at 13 800 g for 10 minutes at 4°C, to purify the protein supernatants from nucleic acids.14 Total protein concentration was determined following the Bio‐Rad protein assay protocol (Bio‐Rad Laboratories; Cat. No. 500‐0006) and used for Western blotting and ELISAs.

Trotta M.C., Ferraro B., Messina A., Panarese I., Gulotta E., Nicoletti G.F., D’Amico M, & Pieretti G. (2019). Telmisartan cardioprotects from the ischaemic/hypoxic damage through a miR‐1‐dependent pathway. Journal of Cellular and Molecular Medicine, 23(10), 6635-6645.

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Western Blot and Immunoprecipitation Protocol

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For cultured cells, the samples were washed by PBS twice after harvest and then digested with RIPA lysis buffer (Sigma) with protease inhibitors (Sigma) and phosphatase inhibitors (Sigma). For mouse tumor samples, the tumors were sliced into small pieces and then ground using tissue grinders in the RIPA lysis buffer (Sigma). After sonication, lysates were collected, and protein concentrations were measured by Pierce™ BCA Protein Assay Kit (ThermoFisher). For western blot, the proteins from each group were mixed with SDS-PAGE loading and boiled for 5 min. For IP, protein samples were mixed with indicated antibodies and rotated for 4 h at 4 °C. Then agarose beads (Santa Cruz) were added to each sample and incubated at 4 °C overnight. The beads were washed with TBST following manual protocol, mixed with SDS-PAGE loading, and boiled for 5 min. Upon transferring to polyvinylidene difluoride membranes, proteins were probed with indicated antibodies. The primary antibodies were diluted in 5% milk in a 1:1000 ratio and incubated at 4°C overnight. Then the membrane was washed 3 times X 5 min with TBST and incubated with diluted second antibodies (1:3000) in the room temperate for 1 h. Before exposure, the membrane was washed 3 times X 5 min with TBST, and then the signal will be detected with the Clarity Western ECL Substrate (BIO-RADs).

Mao F., Kong Y., Liu J., Rao X., Li C., Donahue K., Zhang Y., Jones K., Zhang Q., Xu W, & Liu X. (2022). Diptoindonesin G antagonizes AR signaling and enhances the efficacy of anti-androgen therapy in prostate cancer. The Prostate, 82(8), 917-932.

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Protein Extraction and Analysis for pGSN

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Retro-orbital blood was centrifuged at 2,500rpm for 15min at 4°C. The supernatant was collected and mixed with 1% RIPA lysis buffer (Millipore, MA) in PBS v/v for 2 hours on ice and then centrifuged at 14,000rpm for 20 min. The supernatant was used for protein quantification with the bicinchoninic acid kit (Thermo Fisher Scientific, MA) and for Western blot of pGSN.
Mouse brains were homogenized and proteins were extracted in 1% RIPA lysis buffer (Millipore, MA) in PBS v/v. The resultant suspension was sonicated for 15s and then incubated on ice for 2 hours. The suspensions were centrifuged at 14,000 rpm for 20 min, and used for protein quantification and Western blot of pGSN.

Kevin Li-Chun H., Schob S., Zeller M., Pulli B., Ali M., Wang C., Chiou T.T., Tsang Y.M., Lee P.S., Stossel T.P, & Chen J.W. (2015). Gelsolin decreases actin toxicity and inflammation in murine multiple sclerosis. Journal of neuroimmunology, 287, 36-42.

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Cardiac Ventricle Protein Fractionation

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Frozen fetal cardiac ventricles were used to obtain nuclear and mitochondrial protein fractions for western immunoblot analysis. Briefly, 20–30 mg frozen ventricle pieces were ground with a mortar pestle in liquid N₂ and resuspended in 800 μl of ice-cold homogenization buffer. The nuclear fraction was obtained by using a modified method with two-time homogenization and centrifugation [27 (link)–29 (link)]. The pellet containing the nuclear fraction was resuspended in 1x RIPA Lysis Buffer (Millipore, Calbiochem, MA, USA) and supplemented with 1x protease and phosphatase inhibitor cocktail tablet (Roche Diagnostics, Munich, Germany). The mitochondrial fraction was isolated by a standard differential centrifugation method [11 (link), 13 (link)]. The pellet containing the mitochondrial fraction was resuspended in 1x RIPA Lysis Buffer (Millipore) and supplemented with 1x protease inhibitor cocktail tablet (Roche). All sample protein concentrations were measured by using the Bio-Rad Protein Assay (Bio-Rad). These procedures generate nuclear and mitochondrial fractions [30 (link)] without contamination.

Song H, & Thompson L.P. (2023). Effects of Gestational Hypoxia on PGC1α and Mitochondrial Acetylation in Fetal Guinea Pig Hearts. Reproductive Sciences, 30(10), 2996-3009.

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Protein Expression Analysis via Western Blot

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Cell lysates were prepared using RIPA lysis buffer (Sigma) containing a protease/phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were quantified using the BCA protein assay (Pierce, Appleton, WI, USA). For each cell line, 10 µg of lysate was run in a 4–20% polyacrilamide gradient gel (Bio-Rad, Hercules, CA, USA) and transferred into a nitrocellulose membrane (Bio-Rad). The membrane was blocked in 5% milk in PBS with 0.1% Tween 20 (PBS-T) for 1 h and incubated with the appropriate antibody overnight at 4 °C. FOXC2 (obtained from Dr. Naoyuki Miura) and α-tubulin (Sigma) were used at a 1:1000 dilution. Membranes were washed with PBS-T and incubated with secondary antibodies for 1 h at room temperature, after which they were washed again with PBS-T. The Western blots were scanned and analyzed using an enhanced chemiluminescence (ECL) detection system (Amersham, Amersham, UK) following the protocol provided by the manufacturer. α-tubulin was used as an internal control for protein loading and normalization.

Recouvreux M.S., Miao J., Gozo M.C., Wu J., Walts A.E., Karlan B.Y, & Orsulic S. (2022). FOXC2 Promotes Vasculogenic Mimicry in Ovarian Cancer. Cancers, 14(19), 4851.

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Comparative Analysis of Cancer Cell Lines

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The human colorectal cancer cells (HCT-116), cervical cancer cells (HeLa), lung adenocarcinoma cell line (A549) and human breast cancer cells (MCF7) were bought from American Type Culture Collection (ATCC). The cells were incubated in RPMI-1640 or Dulbecco’s Modified Eagle Medium (DMEM) supplied with 10% foetal bovine serum (FBS) and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin), which were bought from GIBCO. Cell Counting Kit-8 and 4′, 6-Diamidino-2-phenylindole (DAPI) were bought from MCE (Monmouth Junction, NJ, USA). Enhanced chemiluminescence (ECL) kit, Mitochondrial Staining kit and Lysosome Staining Kit were obtained from BD (USA). Protease inhibitor cocktail and RIPA Lysis Buffer were bought from Sigma-Aldrich (Merck KGaA), Bicinchoninic acid (BCA) assay protein kit from EMD Millipore, and PVDF membranes from Bio Basic, Inc., Markham, ON, Canada.

Xie Z., Duo Y., Fan T., Zhu Y., Feng S., Li C., Guo H., Ge Y., Ahmed S., Huang W., Liu H., Qi L., Guo R., Li D., Prasad P.N, & Zhang H. (2022). Light-induced tumor theranostics based on chemical-exfoliated borophene. Light, Science & Applications, 11, 324.

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Western Blot Analysis of p38 Protein

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Cells exposed to different treatments (72 h) (see above) were collected and centrifuged, and total proteins were extracted with RIPA lysis buffer (Sigma-Aldrich, Saint Louis, MO, USA) to determine protein concentration using Bradford. For electrophoresis, 40 µg of proteins from each sample were heated at 95 °C for 5 min and separated in 10% SDS-PAGE gel. Fractions were transferred to a nitrocellulose membrane (45 µm pore size) (Millipore, Burlington, MA, USA), blocked in 5% milk in PBS supplemented with 0.1% Tween-20 (Bio-Rad, Hercules, CA, USA) for 1 h and incubated with the anti-p38 rabbit primary antibody (1:1000) (Cell Signaling Technologies, Barcelona, Spain) overnight at 4 °C. Then, a secondary antibody (Sigma-Aldrich, Saint Louis, MO, USA) was added (1:5000). The membranes were revealed by chemiluminescence (Amersham Biosciences, Saint Louis, MO, USA). β-actin detection (Sigma-Aldrich, Saint Louis, MO, USA) served as an internal control. The gels were analyzed using Quantity One analytical software (Bio-Rad, Hercules, CA, USA).

Doello K., Mesas C., Quiñonero F., Perazzoli G., Cabeza L., Prados J., Melguizo C, & Ortiz R. (2021). The Antitumor Activity of Sodium Selenite Alone and in Combination with Gemcitabine in Pancreatic Cancer: An In Vitro and In Vivo Study. Cancers, 13(13), 3169.

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Chondrogenic Marker Analysis in NSCs

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For further analysis of chondrogenic markers, the NSCs were lysed using RIPA lysis buffer (Sigma-Aldrich). A total of 30 µg protein was subject to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with the use of 10 % resolving gels, then followed by transfer to polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The membrane was incubated with primary antibodies specific for COL II (Abcam, ab34712), SOX9 (Santa Cruz, sc-166,505), Aggrecan (Santa Cruz, sc-70,332), COL I (Abcam, ab34710), and β-actin (Santa Cruz, sc-47,778), respectively, all of which were diluted in 1:1000. Those markers were visualized by chemiluminescence using Western ECL substrate (Thermo Scientific), and the luminescent images were analyzed using a LAS-3000 (Fujifilm).

Noh Y.K., Kim S.W., Kim I.H, & Park K. (2021). Human nasal septal chondrocytes (NSCs) preconditioned on NSC-derived matrix improve their chondrogenic potential. Biomaterials Research, 25, 10.

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Rat Primary Astrocyte Culture Protocol

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Hanks’ balanced salt solution (HBSS) Ca2+ and Mg2+ free ((Gibco, Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA), HBSS with Ca2+ and Mg2+ ((Gibco, Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA), papain (Sigma-Aldrich, St. Louis, MO, USA), DNase (Sigma-Aldrich, St. Louis, MO, USA), DMEM Low glucose (1 g/L) (PanEco, Moscow, Russia), DMEM High glucose (4 g/L) (PanEco, Moscow, Russia), antibiotic-antimycotic ((Gibco, Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA), L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), fetal bovine serum (Invitrogen, Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA), trypsin (Gibco, Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA), Lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA), Griess reagent (Sigma-Aldrich, St. Louis, MO, USA), RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), Complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany), Quantikine Rat Il-6, R&D Systems, USA, 4-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich, St. Louis, MO, USA), Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA).

Abdyeva A., Kurtova E., Savinkova I., Galkov M, & Gorbacheva L. (2024). Long-Term Exposure of Cultured Astrocytes to High Glucose Impact on Their LPS-Induced Activation. International Journal of Molecular Sciences, 25(2), 1122.

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