Ripa lysis buffer

Manufactured by Thermo Fisher Scientific

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RIPA lysis buffer is a cell lysis reagent used for the extraction of proteins from cells and tissues. It contains a mixture of ionic and non-ionic detergents that help solubilize cellular components, allowing for the isolation of proteins for further analysis.

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Radioimmunoprecipitation assay (RIPA) lysis and extraction buffers Modified RIPA lysis buffer RIPA lysis extraction buffer Thermo Scientific™ RIPA Lysis and Extraction Buffer RIPA cell lysis and extraction buffer RIPA cell lysis buffer RIPA Lysis and Extraction Buffer Modified radioimmunoprecipitation assay (RIPA) lysis buffer RIPA western lysis buffer RIPA protein lysis buffer

1 677 protocols using ripa lysis buffer

Western Blot Protocol for Protein Analysis

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The specimens were washed repeatedly with cold PBS (Thermo Fisher Scientific #21,600–069) twice and then immersed in RIPA Lysis buffer (Thermo Fisher Scientific #89,901) which was diluted with a protein inhibitor cocktail 1:100 in 1× RIPA Lysis buffer, cleaved for 15 min then collected the supernatant. The protein concentration was determined by BCA assay (Thermo Fisher Scientific #23,227). After that, the supernatant was mixed with 6X SDS loading buffer, and boiled for 10 min, separate by SDS–PAGE. Lysates were then run on an SDS/10–15% polyacrylamide gel (Bio-Rad) and transferred onto 0.45μm PVDF membranes (Invitrogen), blocked with by 1 × TBST buffer containing 5% skim milk at room temperature for 1 h. The membrane was incubated with the first antibody (1:1,000 dilution) at 4°C overnight, rinsed with TBST, and then incubated with the second antibody (1:8,000 dilution) for 1 h at room temperature, washed by TBST buffer solution at 3 times every 5 min, detected by the ECL reagent (Millipore#WBKLS0500), and directly imaged and digitalized using a BioRad VersaDoc 4,000 imaging system and quantified by the Quantity One software (BioRad, CA, United States).

Tang J., Chen Z., Wang Q., Hao W., Gao W.Q, & Xu H. (2021). hnRNPA2B1 Promotes Colon Cancer Progression via the MAPK Pathway. Frontiers in Genetics, 12, 666451.

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Mouse Hemibrains Protein Extraction

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Frozen samples from the posterior half of the from left mouse hemibrains (including neocortex and hippocampus) were homogenized in detergentcontaining RIPA lysis buffer with phosphatase and protease inhibitors in RIPA lysis buffer (Thermo Scientific; 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS).
Homogenates were separated into soluble and insoluble fractions through centrifugation at 100,000 g for 60 min. A BCA assay was used to analyze the samples; protein (20 μg/lane) was loaded into each well of a 4–12% SDS-PAGE gel and run in 5% MES (20X) running buffer at 200V for 50 min. The gels were then blotted onto a nitrocellulose membrane, using Thermo Fisher iBlot 2 western detection stacks. Blots were incubated for detection of specific target proteins using the corresponding antibody listed in Table 1 for two days at 4°C. The membrane was then washed and incubated in horseradish-peroxidase-conjugated secondary antibody of the appropriate species (1:1000, Santa Cruz Biotechnology) for 60 min at room temperature. Bands were then washed and visualized by enhanced chemiluminescence (PerkinElmer, Boston, MA), analyzed with a quantitative Versadoc XL imaging apparatus (BioRad, Hercules, CA), and analyzed with Quantity One Software. β-Actin (1:3000, Sigma Aldrich) was used as a loading control.

Arner A., Rockenstein E., Mante M., Florio J., Masliah D., Salehi B., Adame A., Overk C., Masliah E, & Rissman R.A. (2018). Increased Vulnerability of the Hippocampus in Transgenic Mice Overexpressing APP and Triple Repeat Tau. Journal of Alzheimer's disease : JAD, 61(3), 1201-1219.

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Protein Extraction and Western Blot Analysis

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The total cellular protein and tissue protein was extracted by RIPA Lysis Buffer (Thermo Fisher Scientific, MA, USA) and RIPA Lysis Buffer (Thermo Fisher Scientific) containing protease inhibitors and phosphatase inhibitors (Thermo Fisher Scientific). The protein concentrations of the cell lysates were measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) and equalized before loading. Equal amount of protein extracts from HCC cells or tissues were separated by SDS–PAGE, and transferred onto polyvinylidene fluoride membranes (Sigma-Aldrich, MO, USA). Immunoblot analyses were carried out using the appropriate antibodies, and the bands were visualized using an SuperSignal™ West Pico PLUS chemiluminescence Substrate (Thermo Fisher Scientific).

Jiang Y., Chen S., Li Q., Liang J., Lin W., Li J., Liu Z., Wen M., Cao M, & Hong J. (2021). TANK-Binding Kinase 1 (TBK1) Serves as a Potential Target for Hepatocellular Carcinoma by Enhancing Tumor Immune Infiltration. Frontiers in Immunology, 12, 612139.

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Western Blot Analysis of Signaling Proteins

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Tissues were homogenized and lysed with RIPA lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Similarly, cells were lysed with RIPA lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Protein concentrations of whole-cell lysates were determined using Lowry assays (Bio-Rad). 20 μg of total protein were prepared in LDS sample buffer (Invitrogen), separated on denaturing Bis-Tris gel (Invitrogen), and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked in 5% milk in 0.1% Tween 20 Tris-buffered saline (TBST) and subsequently incubated with primary antibodies against phospho-p44/42 MAPK (pErk1/2; Thr202/Tyr204), total ERK, RasG12D, and P53 (Cell Signaling Technology) in TBST overnight at 4°C, or with β Actin HRP-conjugated for 1 hour at room temperature. Secondary antibodies were purchased from Jackson ImmunoResearch. ECL-Plus substrate (Bio-Rad) and Bio-Rad ChemiDoc MP imager were used according to the manufacturer’s recommendations.

Lasse-Opsahl E., Baliira R., Barravecchia I., McLintock E., Lee J.M., Ferris S.F., Espinoza C.E., Hinshaw R., Cavanaugh S., Robotti M., Brown K., Donahue K., Abdelmalak K.Y., Galban C.J., Frankel T.L., Zhang Y., di Magliano M.P, & Galban S. (2024). WITHDRAWN: Oncogenic KRAS G12D extrinsically induces an immunosuppressive microenvironment in lung adenocarcinoma. bioRxiv.

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Quantifying Amyloid-beta Levels

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Aβ levels were detected by performing immunopreci-pitation of conditioned media before western blot analysis. Therefore, protein A-beads (Amersham Biosciences) and 4G8 antibody (Covance) were used to immunoprecipitate Aβ. After incubating the supernatant with 4G8 antibody overnight at 4°C, the protein A-beads was added into the mixture and further incubated for 6 h at 4°. Following centrifugation, the beads were rinsed with RIPA lysis buffer (Thermo fisher scientific) for five times and then resuspended with the RIPA lysis buffer. Aβ was then immunoblotted by the 6E10 antibody (BioLegend) in Western blot assay.

Hu T., Zhou Y., Lu J., Xia P., Chen Y., Cao X, & Pei G. (2020). A novel rhamnoside derivative PL402 up-regulates matrix metalloproteinase 3/9 to promote Aβ degradation and alleviates Alzheimer’s-like pathology. Aging (Albany NY), 12(1), 481-501.

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Immunoprecipitation of GFP-CsPLA2 and Myc-TM7SF3

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We transiently over-expressed GFP-tagged CssPLA2 and Myc-tagged TM7SF3 in 293T cells for 24 h. Transfected 293T cells were lysed using RIPA lysis buffer (Gibco, USA) and incubated with an anti-PLA2 antibody and protein A-agarose beads (Pierce, USA) overnight at 4 °C. After washing five times with RIPA lysis buffer (Gibco, USA), the beads were eluted with 2× SDS sample buffer and boiled for 8 min at 100 °C. The samples were then analysed by western blot using anti-Myc antibodies (Proteintech, USA).

Wu Y.J., He Q., Shang M., Yin Y.X., Li Y., Du X, & Li X.R. (2021). The NF-κB signalling pathway and TM7SF3 contribute to liver fibrosis caused by secreted phospholipase A2 of Clonorchis sinensis. Parasites & Vectors, 14, 152.

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Comprehensive Protein Expression Analysis

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Bone tissues were crushed and then lysed in RIPA Lysis Buffer (Invitrogen), BMSCs were also lysed in RIPA Lysis Buffer (Invitrogen), and the extracted proteins were quantified by BCA protein assay reagent (Thermo Fisher Scientific Inc). Proteins (30 μg) were separated by SDS-PAGE before being transferred onto polyvinylidene difluoride membranes. The membranes were blocked by 5% bovine serum albumin, and probed with primary antibodies: anti-MeCP2 and anti-GAPDH (1:2000; Abcam, Cambridge, MA, USA), anti-ALP and anti-RUNX2 (1:2500; Abcam), anti-COL1A1 and anti-OCN (1:3000; Abcam), anti-FOXF1 and anti-Wnt5a (1:3500; Abcam), anti-β-Catenin and anti-APC (1:4000; Abcam). The membrane was washed by phosphate Buffered Saline containing Tween 20 buffer and then incubated with corresponding goat anti-mouse or goat anti-rabbit secondary antibodies (1:4500; Abcam), and the protein strips were visualized by Colorimetric Western blotting Kit (Sigma-Aldrich; St. Louis, MO, USA) according to published work [20 (link)].

Ji W, & Sun X. (2021). Methyl-CpG-binding protein 2 promotes osteogenic differentiation of bone marrow mesenchymal stem cells through regulating forkhead box F1/Wnt/β-Catenin axis. Bioengineered, 13(1), 583-592.

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Oridonin Modulates Stress Response Proteins

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WI-38 cells were first induced with 1 μM doxorubicin for 12 h, and then treated with oridonin or DMSO-containing medium for 48 h. WI-38 cells homogenized in RIPA lysis buffer (Thermal Scientific) with protease inhibitors (Roche) and 1% phosphatase inhibitors (Applygen). The protein extracts were homogenized using a Dounce homogenizer and spun at 12,000 g for 20 min at 4°C. BCA analysis (Pierce) was used for protein quantification, and then SDS-PAGE was used to separate the proteins and then transfer to a PVDF membrane (Amersham Biosciences). The botting membranes were incubated with primary antibodies against ACTIN, p-FOXO1, FOXO1, p21, p53, IL-1α or IL-1β (ProteinTech), IL-6 (ABclonal), and IL-8 (Immunoway) overnight at 4°C. Secondary goat anti-rabbit or anti-mouse IgG (Scicrest) was added for blotting. ECL Plus kit (Amersham Biosciences) was used for visualization. The optical density of each band was used to quantify relative protein level.

An Y., Zhu J., Wang X., Sun X., Luo C., Zhang Y., Ye Y., Li X., Abulizi A., Huang Z., Zhang H., Yang B, & Xie Z. (2022). Oridonin Delays Aging Through the AKT Signaling Pathway. Frontiers in Pharmacology, 13, 888247.

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Carbamylation Protein Isolation Protocol

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Tissues were homogenised in RIPA lysis buffer (Thermal Scientific) containing protease inhibitor cocktail (Roche). The extract was homogenised and spun at 12,000 g for 20 min at 4 °C. Pre-cleared lysates were incubated with anti-carbamylation antibody (Abcam, ab175132) coupled to protein A/G agarose (Santa Cruz) overnight at 4 °C. The immune complex was collected by centrifugation, and the pellets were washed in lysis buffer three times for 10 min with rotation at 4 °C. Carbamylated proteins were washed with cold lysis buffer six times, resuspended in SDS sample buffer, and subjected to SDS-PAGE and Western blot analysis.

Wang H., Huang B., Wang W., Li J., Chen Y., Flynn T., Zhao M., Zhou Z., Lin X., Zhang Y., Xu M., Li K., Tian K., Yuan D., Zhou P., Hu L., Zhong D., Zhu S., Li J., Chen D., Wang K., Liang J., He Q., Sun J., Shi J., Yan L., Sands J.M., Xie Z., Lian X., Xu D., Ran J, & Yang B. (2019). High urea induces depression and LTP impairment through mTOR signalling suppression caused by carbamylation. EBioMedicine, 48, 478-490.

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Quantitative Histone Acetylation Analysis

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WI‐38 and 2BS cells were homogenized in RIPA lysis buffer (Thermal Scientific) supplemented using a protease inhibitor cocktail (Roche). A Dounce homogenizer was used to homogenize these protein extracts prior to spinning for 20 min at 12,000 g at 4°C. Total protein extraction kits for microbes with thick cell walls (Invent) were used when extracting protein from yeast. A BCA assay (Pierce) was used for protein quantification, after which proteins were separated with SDS‐PAGE gels and transferred onto PVDF membranes (Amersham Biosciences). Blots were then probed using polyclonal antibodies against H3 (Active motif), H3‐Ac (Active motif), H4 (Active motif), H4‐Ac (Active motif), H3K9Ac (Active motif), H3K14Ac (Active motif), H3K18Ac (Active motif), H3K23Ac (Active motif), H3K36Ac (Active motif), Ac‐lysine (Active motif), and β‐tublin (Proteintech). Secondary goat anti‐rabbit or anti‐mouse IgG (Scicrest) was then used to probe blots, which then underwent development using an ECL Plus Kit (Amersham Biosciences). The optical density of each band was used as a means of quantifying relative protein levels.

Huang B., Zhong D., Zhu J., An Y., Gao M., Zhu S., Dang W., Wang X., Yang B, & Xie Z. (2020). Inhibition of histone acetyltransferase GCN5 extends lifespan in both yeast and human cell lines. Aging Cell, 19(4), e13129.

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