Ripa lysis buffer

Manufactured by Elabscience

Sourced in China

RIPA lysis buffer is a commonly used reagent in cell and tissue lysis. It contains a combination of ionic and non-ionic detergents that facilitate the disruption of cell membranes and the solubilization of cellular proteins. The buffer also includes other components to maintain the stability and activity of the extracted proteins.

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Lab products found in correlation

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Close spelling variants of this product

RIPA buffer (5 citations)

18 protocols using ripa lysis buffer

Evaluating CD38 and Sirt6 Expression in Myocardial Cells

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H9c2 cells were cultured and collected by centrifugation. RIPA lysis buffer (Elabscience, China) was used to lyse the samples. Total protein was separated by electrophoresis with 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to PVDF membranes. Anti-CD38 (ABclonal, China), anti-Sirt6 (CST, USA) and anti-β-actin antibodies (Elabscience, China) were incubated with the membranes. β-actin expression was used as an internal control. The relative expression levels of CD38 and Sirt6 were quantified using ImageJ software.
CD38 expression and Sirt6 expression in mouse myocardial tissues were detected by a similar protocol. The heart tissues were collected and lysed with RIPA lysis buffer (Elabscience, China) to extract total protein. A Pierce TM BCA protein assay kit (Thermo, USA) was used to quantify protein.

Zhou H., Liu S., Zhang N., Fang K., Zong J., An Y, & Chang X. (2022). Downregulation of Sirt6 by CD38 promotes cell senescence and aging. Aging (Albany NY), 14(23), 9730-9757.

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Quantitative Western Blot Analysis

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PRKs were lysed using RIPA lysis buffer (Elabscience Biotechnology, Inc.). Lysate protein concentrations were determined using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.) and resolved denatured proteins (20 µg) using 10% SDS-PAGE (Elabscience Biotechnology, Inc.). Protein bands were transferred onto a PVDF membrane at 60 V for 2 h at 4˚C. Then, Ponceau S dye was used to stain the membrane at 25˚C for 5 min. The membrane was blocked with 5% BSA at 25˚C for 2 h, incubated with primary antibodies (anti-IGF1R, anti-PI3K, anti-AKT, anti-p-PI3K, anti-p-AKT, anti-eNOS, anti-p-eNOS) overnight at 4˚C, rinsed with TBS-0.05%Tween 20 buffer (Beijing Solarbio Science & Technology Co., Ltd.) twice, for 10 min each time, and incubated with the secondary antibody (Goat anti-rabbit; Horseradish peroxidase) for 1.5 h at 23±2˚C. Details of the antibodies are shown in Table II. Subsequently, ECL reagent (Thermo Fisher Scientific, Inc.) was used for the chemiluminescence reaction. Finally, the membrane was developed and fixed using a Developer and Fixer Kit (Beyotime Institute of Biotechnology), and the densitometry was quantified using ImageJ software (version 1.8.0; National Institutes of Health, Bethesda, MD, USA).

Mai H., Huang Z., Zhang X., Zhang Y., Chen J., Chen M., Zhang Y., Song Y., Wang B., Lin Y, & Gu S. (2022). Protective effects of endothelial progenitor cell microvesicles carrying miR‑98‑5p on angiotensin II‑induced rat kidney cell injury. Experimental and Therapeutic Medicine, 24(5), 702.

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Protein Expression Analysis in Heart Tissue

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The cells or extracted heart tissue samples were homogenized using RIPA lysis buffer (Elabscience) and centrifuged at 15,000 × g for 10 min at 4 °C. The supernatant was collected, and the protein concentration was determined using a BCA assay kit (Thermo Fisher Scientific). The proteins were separated using 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Merck Millipore, USA). The membranes were blocked with 5% skim milk for 2 h and incubated overnight at 4 °C with the primary antibodies against the following proteins: TFPI2 (1:1000, ab186747; Abcam), iNOS (1:1000, ab129372; Abcam), Arg-1 (1:1000, ab91279; Abcam), PPAR-γ (1:1000, Santa Cruz), collagen I (1:50, ab138492, Abcam), collagen III (1:50, ab184993, Abcam), MMP2 (1:50, ab92536, Abcam), MMP9 (1:50, ab283575, Abcam), and β-actin (1:4000, 8H10D10, CST). Horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG (1:10,000, Absin, Shanghai, China) was used as the secondary antibody and incubated for 1 h at room temperature. Target bands were visualized using chemiluminescent ECL (Merck Millipore, USA) and detected using an Amersham Imager 600 (GE Healthcare, Little Chalfont, UK). The images were analysed using the ImageJ data acquisition software.

Guo M., Xia Z., Hong Y., Ji H., Li F., Liu W., Li S., Xin H., Tan K, & Lian Z. (2023). The TFPI2–PPARγ axis induces M2 polarization and inhibits fibroblast activation to promote recovery from post-myocardial infarction in diabetic mice. Journal of Inflammation (London, England), 20, 35.

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Protein Extraction and Western Blot Analysis

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The proteins extraction was prepared from H9c2 cells [20 (link)]. Then, proteins were lysed in the RIPA lysis buffer (Elabscience, Wuhan, China) on ice. The quantitation of proteins concentration was conducted with the aid of the BCA Protein Assay kit (Beyotime, Shanghai, China) in the light of the guidance of manufacturer. Subsequently, the protein samples were separated by SDS-PAGE gels, followed by the shifts onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After washing with PBS and blocking with 5% skimmed milk, primary antibodies (SFRP4, 1:1000, ab154167; IL-1β, 1:1000, ab254360; TNF-α, 1:1000, ab215188; PTPN12, 1:1000, ab289859; PI3K, 1:1000, ab191606; AKT, 1:1000, ab179463; p-PI3K, 1:1000, ab182651; p-AKT, 1:1000, ab192623; GAPDH, 1:1000, ab8245) were put in the incubator and cultivated with these membranes overnight at room temperature, followed by an incubation of secondary antibodies labeled by horseradish peroxidase at indoor temperature for 1 h. The detection of protein bands was implemented through Image J software (National Institutes of Health, Bethesda, MD, USA).

Bai Z, & Hao X. (2022). Downregulation of secreted frizzled-related protein 4 inhibits hypoxia/reoxygenation injury in diabetic cardiomyocytes by protein tyrosine phosphatase nonreceptor type 12. Bioengineered, 13(3), 7697-7708.

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Protein Extraction and Western Blot Analysis

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Total protein was extracted from cells, lung tissues of patients, and xenograft tumour tissues of mice using RIPA lysis buffer (Elabscience). Total protein was subjected to 10% SDS-PAGE, and then transferred to polyvinylidene difluoride membranes. Polyvinylidene difluoride membranes were blocked with 5% non-fat milk in TBST for 1 h at 37 °C. After blocking, the membrane was incubated with primary antibodies (CHCHD4, cat no. 21090-1-AP, Proteintech, Wuhan, China; Bcl-2, cat no. 26593-1-AP, Proteintech; Cleaved-PARP, cat no. #5625, Cell Signalling, USA; Bax, cat no. 50599-2-Ig, Proteintech; E-cadherin, cat no. A20798, Abclonal, Wuhan, China; N-cadherin, cat. ab280375, Abcam, USA; vimentin, cat no. 10366-1-AP, Proteintech; MYC, cat no. 10828-1-AP, Proteintech, USA; Snai1, cat no. 13099-1-AP, Proteintech; Snai2, cat no. 12129-1-AP, Proteintech; and Twist1, cat no. A7314, Abclonal,; GAPDH, cat no. 10494-1-AP, Proteintech) overnight at 4 °C. Membranes were then incubated with horseradish peroxidase-conjugated goat polyclonal anti-rabbit IgG secondary antibodies (cat. no. ab150077; Abcam) for 1 h at room temperature. The target bands were visualised using a chemiluminescence kit (Beyotime).

Zhou Y., Zhao Y., Ma W., Zhang L., Jiang Y, & Dong W. (2022). USF1-CHCHD4 axis promotes lung adenocarcinoma progression partially via activating the MYC pathway. Discover. Oncology, 13, 136.

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Western Blot Analysis of Protein Extraction

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Total protein from 16HBE cells was extracted using RIPA lysis buffer (Elabscience, Wuhan, China). After protein concentrations were determined by BCA Protein Assay Kit (Takara, Japan), equal amount of protein for each sample was resolved with SDS-PAGE (10%) and then immobilized onto PVDF membranes (Roche, Basel, Switzerland). The membranes were soaked in 5% non-fat dried milk in PBS and incubated with antibodies against MUC5AC (1:1,000, ab24071, Abcam), p65 (0.5 µg/mL, ab16502, Abcam), Lamin B1 (0.1 µg/mL, ab16048, Abcam), and β-actin (1:5,000; ab8226, Abcam) overnight at 4 °C respectively, and then incubation with corresponding secondary antibody at room temperature for 60 min. The blots were revealed with an ECL kit (Abcam) and analyzed by Image Software (NIH, Bethesda, MD, USA).

Fu H.T., Zhang Y., Zhang P., Wu H., Sun X.Q., Shen S.Y, & Dou D.B. (2021). Tumor necrosis factor-α promotes airway mucus hypersecretion by repressing miR-146a-5p and miR-134-5p levels in human airway epithelial cells. Translational Cancer Research, 10(9), 4047-4056.

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RNA Immunoprecipitation of circZDHHC20 and miR-144

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HTR-8/SVneo cells were homogenized in RIPA lysis buffer (Elabscience, Wuhan, China) supplemented with protease inhibitor cocktails (Thermo Fisher Scientific). Cell lysates were incubated with magnetic bead-coupled anti-Argonaute2 antibody (anti-Ago2, Abcam, Cambridge, UK) or a negative control IgG antibody (Abcam), following the instructions of the Magna RNA Immunoprecipitation Kit (Millipore, Zug, Switzerland). Total RNA was isolated from the beads and subjected to qRT-PCR for the enrichment of circZDHHC20, miR-144, and GRHL2.

Zhou B., Zhang X., Li T., Xie R., Zhou J., Luo Y, & Yang C. (2020). CircZDHHC20 represses the proliferation, migration and invasion in trophoblast cells by miR-144/GRHL2 axis. Cancer Cell International, 20, 19.

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Western Blot Analysis of Autophagy Proteins

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Cellular and ovarian lysates were prepared using RIPA lysis buffer (Elabscience, Wuhan, China), with the addition of protease and phosphatase inhibitors. Protein concentrations were quantified using the BCA Protein Concentration Assay Kit (Beyotime, Shanghai, China). Following this, 20 μg of protein was subjected to separation via SDS-PAGE and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Boston, USA). The membranes were blocked in 5% skimmed milk for 2 h and probed with antibodies against SIRT1(proteintech, 13161-1-AP, 1:1000), ATG14 (proteintech, 19491-1-AP, 1:1000), Beclin1 (CST, 3495, 1:1000), P62/SQSTM1 (CST, 39749, 1:1000), ATG5 (CST, 12994, 1:1000) and GAPDH (proteintech, 60004-1-Ig, 1:10000) overnight at 4 °C. Subsequently, the membranes were exposed to secondary antibodies for 1 h at room temperature. Protein bands were visualized utilizing the ECL method, and the intensity of the bands was quantified using Image J (NIH, USA).

Luo C., Wei L., Qian F., Bo L., Gao S., Yang G, & Mao C. (2024). LncRNA HOTAIR regulates autophagy and proliferation mechanisms in premature ovarian insufficiency through the miR-148b-3p/ATG14 axis. Cell Death Discovery, 10, 44.

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Western Blot Analysis of Protein Expression

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Total proteins were isolated from cells using RIPA Lysis Buffer (Elabscience, Wuhan, China), and then quantified using a BCA Protein Assay Kit (ThermoFisher, SanJose, CA, USA). The protein samples were separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked in 5.0% non-fat milk for 45 min and incubated with primary antibody at 4°C overnight. The primary antibodies included anti-GAPDH (1:1000, ab9485, Abcam, Cambridge, England), -PI3K (1:1000, 4292, CST, Danvers, MA, USA), -p-PI3K (1:1000, 17,366, CST), -Akt (1:1000, 9272, CST), -p-Akt (1:1000, 4060, CST), -MMP-2 (1:1000, ab97779, Abcam), -MMP-9 (1:1000, ab38898, Abcam,), -Bcl-2 (1:1000, ab32124, Abcam), and -Bax (1:1000, ab32503, Abcam). Subsequently, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000, G-21234, Invitrogen) for 1 h at 25°C. Protein bands were visualized using a Chemiluminescent Substrate Kit (Bio-Rad, Hercules, CA, USA). GAPDH was used as the internal control.

Ma F., An K, & Li Y. (2020). Silencing of Long Non-Coding RNA-HCG18 Inhibits the Tumorigenesis of Gastric Cancer Through Blocking PI3K/Akt Pathway. OncoTargets and therapy, 13, 2225-2234.

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Western Blot Analysis of DNA Damage Markers

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Protein was extracted with RIPA lysis buffer (Elabscience Biotechnology). Protein samples were resolved by 10% SDS‐PAGE gels and transferred onto PVDF membranes (Millipore). After being blocked with skimmed milk, the membranes were incubated with anti‐γH2AX (1:1000, Abcam), and anti‐THBS2 (1:1000, Boster) or anti‐GAPDH (1:2000, Boster). After further incubatation with horseradish peroxidase (HRP) conjugated goat anti‐rabbit/mouse IgG (H + L) (1:5000, Boster), the protein signal was detected by enhanced chemiluminescence (Millipore).

Yang Z., Wu H., Zhang K., Rao S., Qi S., Liu M., Chen Y, & Wang Y. (2022). Circ_0007580 knockdown strengthens the radiosensitivity of non‐small cell lung cancer via the miR‐598‐dependent regulation of THBS2. Thoracic Cancer, 13(5), 678-689.

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