Ripa lysis

Manufactured by Thermo Fisher Scientific

Sourced in United States, China

RIPA lysis is a protein extraction buffer used to lyse cells and solubilize proteins. It contains a combination of detergents, salts, and buffers that help to disrupt cell membranes and release proteins from the cellular environment.

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Close spelling variants of this product

Modified radioimmunoprecipitation assay (RIPA) lysis buffer (2 citations) RIPA protein lysis buffer (8 citations) Pierce RIPA cell lysis buffer (4 citations) RIPA) lysis buffer solution (2 citations) Pierce RIPA lysis buffer (35 citations) RIPA cell lysis and extraction buffer (2 citations) RIPA lysis extraction buffer (8 citations) Modified RIPA lysis buffer (2 citations) Thermo Scientific™ RIPA Lysis and Extraction Buffer (2 citations) Radio Immunoprecipitation Assay (RIPA) lysis buffer (23 citations)

24 protocols using ripa lysis

Comprehensive Protein Analysis Protocol

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RIPA lysis (ThermoFisher, USA) was used for extracting total cell protein. Protein concentration was tested by BCA assay (ThermoFisher, USA). Equal amounts of protein were separated by SDS-PAGE gel as regular protocol. And the protein was transferred from gel to a PVDF membrane (Millipore). Then the PVDF membrane was blocked in 5% BSA-TBST (Sigma, USA) for 2 h at room temperature, followed by primary antibody at 4-degree overnight. The membrane was washed 10 min 3 times with TBST, followed by secondary antibody at room temperature for 1 h. After another washing cycle, the membrane was visualized by ultra-sensitive ECL kit. Results was calculated by Target protein/ β-actin based on band intensity tested by ImageJ.
The related antibodies used were as follows:
E-cadherin (CST, Boston, USA,14472), N-cadherin (CST, Boston, USA, 13116), Vimentin (CST, Boston, USA, 5741), Snail (CST, Boston, USA), MMP9(CST, Boston, USA), smad2/3((abcam, USA, ab202445), p-smad2/3 (CST, Boston, USA),BMP4 (abcam, USA, ab124715),P-SMAD1/5/8(CST, Boston, USA, 13820), Anti-SMAD1/5/8 antibody (abcam, USA,ab80255), and TGF-BETA1 (abcam, USA, ab179695), SMAD6(SANTA CRUZ, USA, sc-25,321).

Liu L., Zhang C., Wang J., Liu X., Qu H., Zhang G., Liang T., Wang J, & Zhang J. (2021). A high level of lncFGD5-AS1 inhibits epithelial-to-Mesenchymal transition by regulating the miR-196a-5p/SMAD6/BMP axis in gastric Cancer. BMC Cancer, 21, 453.

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Protein Expression Analysis using Western Blot

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The cultured cells were harvested to extract total proteins with RIPA lysis (Thermo Scientific, USA, and their concentration was detected using bicinchoninic acid method (Thermo Scientific, USA), then adjusted to 4μg/μL. Following separating with 12% SDS-PAGE, the proteins were transferred to a polyvinylidene difluoride membrane. Subsequently, the membrane was dyed in Ponceau S working solution, soaked in PBST for 5 min and washed, sealed with 5% non-fat milk powder for 2 h, and incubated overnight at 4° C along with primary antibodies against CD9 and CD63, MMP-14 (1: 1000, ABCM, USA). After washing the membrane to remove the primary antibodies, horseradish peroxidase labeled goat anti-rabbit (Abcam, USA) secondary antibody (1: 5000) was added for another 1-h incubation (37° C). Afterwards, the membrane was washed 3 times with PBS for 5 min each, and the excess liquid was dried with a filter paper. The development was performed with enhanced chemiluminescence (ECL) in a darkroom. Gray values of luminescent protein bands were measured with Quantity One. Relative expression of target proteins = gray value of target protein band /gray value of β-Actin protein band.

Chang L., Gao H., Wang L., Wang N., Zhang S., Zhou X, & Yang H. (2021). Exosomes derived from miR-1228 overexpressing bone marrow-mesenchymal stem cells promote growth of gastric cancer cells. Aging (Albany NY), 13(8), 11808-11821.

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Rosemary Extract and Cannabinoid Derivatives

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The ethanolic extract of Rosemary was kindly provided by Dr Yiming Li from Shanghai University of Traditional Chinese Medicine. CS, with a purity of >98%, was purchased from Shanghai NatureStandard Biotechnology Co., LTD (Shanghai, P.R. China). DCS and DCSD were synthesized from the structural modification of CS. Dulbecco's modified Eagle's minimal essential medium (DMEM) (high glucose), RMPI‐1640, penicillin/streptomycin, and trypsin/EDTA were purchased from Hyclone (Los Angeles, CA, United States). Horse serum was purchased from Gibco (New York, NY, United States). Foetal bovine serum (FBS) was derived from Biological Industries (Kibbutz Beit Haemek, Israel). RIPA Lysis, Halt Protease, and Phosphatase Inhibitor Cocktail (100×) were purchased from Thermo Scientific (Rockford, IL, United States). BCA protein assay kit used to quantify protein concentration was purchased from Beyotime (Shanghai, P.R. China). TNF‐α and IL‐6 were purchased from PeproTech (Rocky Hill, CT, United States). Other chemicals, except where specially noted, were purchased from Sigma‐Aldrich Chemical Co. (St. Louis, MO, United States).

Lu S., Li Y., Shen Q., Zhang W., Gu X., Ma M., Li Y., Zhang L., Liu X, & Zhang X. (2021). Carnosol and its analogues attenuate muscle atrophy and fat lipolysis induced by cancer cachexia. Journal of Cachexia, Sarcopenia and Muscle, 12(3), 779-795.

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Western Blot Analysis of Adipocyte Proteins

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The 3T3-L1 adipocytes were lysed with ice-cold RIPA Lysis (Thermo Fisher Scientific, Shanghai, China) and incubated on ice for 30 min to extract proteins. The supernatant was collected by centrifugation at 14,000× g for 15 min at 4 °C for further testing. The protein concentration of each sample was measured by the BCA Protein Assay Kit (Thermo Fisher Scientific, Shanghai, China). Then the supernatant was boiled in 5× loading buffer for 10 min and electrophoresed on a 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The protein bands were transferred to an NC membrane (Millipore, Burlington, MA, USA) and blocked with 5% (w/v) skim milk for 1 h at room temperature. After three washes in Tris-buffered saline containing Tween 20 (TBST), the membranes were incubated with designated primary antibodies (p-Akt, Akt, GLUT4) at 4 °C overnight then washed again with TBST 5 times, and the secondary antibody conjugated with peroxidase was incubated at room temperature with 1:5000 dilution. After washing the membrane, protein was detected using ECL (Millipore, Burlington, NJ, USA) with GAPDH as a loading reference [37 (link)].

Yi G., Sang X., Zhu Y., Zhou D., Yang S., Huo Y., Liu Y., Safdar B, & Bu X. (2023). The SWGEDWGEIW from Soybean Peptides Reduces Insulin Resistance in 3T3-L1 Adipocytes by Activating p-Akt/GLUT4 Signaling Pathway. Molecules, 28(7), 3001.

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Western Blot Analysis of AKT and PI3K Signaling

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Following IL-1β treatment or transfection, the total proteins of C28/I2 cells were extracted using RIPA lysis (Thermo Fisher Scientific, Inc.) and the protein content was evaluated using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Proteins (30 µg per lane) were separated by electrophoresis on 15% SDS-PAGE gels and transferred to polyvinylidenedifluoride membranes. Afterwards, membranes were blocked at room temperature for 1 h with 5% skimmed milk powder, and then incubated with primary antibodies against AKT (cat. no. #4685), p-AKT (cat. no. #4060), PI3K (cat. no. #4249) and p-PI3K (cat. no. #17366) (1:1,000 dilution; all from Cell Signaling Technology, Inc.) overnight at 4˚C. After being washed with phosphate-buffered saline three times for 5 min, they were incubated with goat anti-rabbit IgG H&L (HRP) secondary antibodies (cat. no. #7074; 1:2,000 dilution; Cell Signaling Technology, Inc.) for 1 h at room temperature according to the manufacturer's instructions. Protein bands were visualized by enhanced chemiluminescence (Beyotime Institute of Biotechnology). Densitometric measurements were performed using ImageJ version 1.48 Software (National Institutes of Health).

Xie W., Jiang L., Huang X., Shang H., Gao M., You W., Tan J., Yan H, & Sun W. (2021). lncRNA MEG8 is downregulated in osteoarthritis and regulates chondrocyte cell proliferation, apoptosis and inflammation. Experimental and Therapeutic Medicine, 22(4), 1153.

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Protein Expression Analysis via Western Blot

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The treated cells were homogenized on an ice-cold RIPA Lysis (Thermo Fisher Scientific, Waltham, MA, USA) and quantified using a BCA assay kit (Pierce, Santa Cruz, CA, USA). The proteins were fractionated using SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes, blocked with 5% nonfat milk in TBST, and then blotted with specific antibodies at room temperature overnight. The antibodies used were as follows: anti-YTHDF1 (1:400, Proteintech 17479-1-AP), anti-CCNB1 (1:500, Santa Cruz Biotechnology sc-245), and anti-GAPDH (1:2000, Santa Cruz Biotechnology sc-47724). The bands were visualized using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Waltham, MA, USA).

Lou X., Ning J., Liu W., Li K., Qian B., Xu D., Wu Y., Zhang D, & Cui W. (2021). YTHDF1 Promotes Cyclin B1 Translation through m6A Modulation and Contributes to the Poor Prognosis of Lung Adenocarcinoma with KRAS/TP53 Co-Mutation. Cells, 10(7), 1669.

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Cardiac Fibroblast Protein Expression

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The proteins of heart tissue or cardiac fibroblasts were prepared in RIPA lysis (Thermo fisher Scientific, USA). The following antibodies including CREG (Abcam), αSMA (Abcam), collagen-1 (Abcam), cleaved caspase 3 (Cell Signaling Technology, USA), Bax (Cell Signaling Technology, USA), proliferating cell nuclear antigen (PCNA, Abcam), CDC42 (Abcam), Rac1 (Abcam) and RhoA (Abcam), p21-activated kinase 1 (PAK1, Cell Signaling Technology), phosphorylated-PAK1 (Abcam) and insulin-like growth factor-2 receptor (IGF2R, Abcam) were used. β-actin was as the internal reference (Abcam).

Liu D., Tian X., Liu Y., Song H., Cheng X., Zhang X., Yan C, & Han Y. (2021). CREG ameliorates the phenotypic switching of cardiac fibroblasts after myocardial infarction via modulation of CDC42. Cell Death & Disease, 12(4), 355.

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ALP Enzymatic Activity Assay

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RIPA Lysis, extraction buffer and p-nitrophenyl phosphate (p-NPP) (Thermo Fisher, Waltham, MA, USA) were used to perform the ALP assay. pNPP solution used to lyse the substrate was incubated with cell lysate. ALP in cell lysate converted pNPP substrate to p-nitrophenol. A standard curve of Optical Density (OD) plotted against 4-nitrophenol concentration was generated and used in estimation of ALP activity. By comparing the OD obtained from reaction mixture to standard curve of 4-nitrophenol, the quantity of pNPP substrate converted to p-nitrophenol over time was estimated.

Ng K., Azari P., Nam H.Y., Xu F, & Pingguan-Murphy B. (2019). Electrospin-Coating of Paper: A Natural Extracellular Matrix Inspired Design of Scaffold. Polymers, 11(4), 650.

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Protein Analysis of NSCLC Cells

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Protein samples were prepared from NSCLC cells using RIPA lysis (Thermo Fisher Scientific); the total protein concentration was extracted with extraction buffer (Thermo Fisher Scientific) and electrophored with 100 V SDS‐PAGE. Then the protein samples were quantified by Bradford method before transferred to PVDF membrane. After being blocked with 10% goat serum for 0.5 h, the membrane was probed with primary antibodies against VIM, Bax, Bcl‐2, Caspase‐3, P53, p‐JAK2, JAK2, p‐STAT3, STAT3, β‐actin, and GAPDH at 4°C. Then the membranes were subjected to secondary antibody (diluted at 1:5000) at room temperature for 2 h. Chemiluminesence Western blot reagents (Thermo Fisher Scientific) and ECL System (GE Healthcare) were applied to observe the protein band. The band intensity in protein imprinting was determined by ImageJ software.

Wang B., Li J., Li Y., Liang T, & Chu X. (2022). MiR‐630 suppresses non‐small cell lung cancer by targeting vimentin. Journal of Clinical Laboratory Analysis, 36(9), e24536.

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Cell Culture Reagents Sourcing

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Penicillin/streptomycin, DMEM medium (high glucose), RPMI-1640 medium, and trypsin/EDTA were provided by Hyclone (Los Angeles, CA, United States). Carnosol was provided by NatureStandard Co., LTD. (Shanghai, P.R. China). FBS was purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Halt Protease, and Phosphatase Inhibitor Cocktail (×100) and RIPA Lysis were obtained from Thermo Scientific (Rockford, IL, United States). DAPI and BCA protein quantification kit were purchased from Beyotime (Hangzhou, P.R. China). And most of the other chemicals were provided by Sigma-Aldrich Chemical Co., (St. Louis, MO, United States).

Fang Q.Y., Wang Y.P., Zhang R.Q., Fan M., Feng L.X., Guo X.D., Cheng C.R., Zhang X.W, & Liu X. (2024). Carnosol ameliorated cancer cachexia-associated myotube atrophy by targeting P5CS and its downstream pathways. Frontiers in Pharmacology, 14, 1291194.

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