Nonidet p 40 lysis buffer

Manufactured by Merck Group

Sourced in United States

Nonidet P-40 lysis buffer is a non-ionic detergent used for cell lysis and protein extraction in various laboratory applications. It disrupts cell membranes to release cellular contents, including proteins, for further analysis or purification.

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Lysis buffer (324 citations) Nonidet P-40 (259 citations)

4 protocols using nonidet p 40 lysis buffer

Immunoprecipitation of RB or Necrosome Complexes

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For RB or necrosome immunoprecipitation, cells were seeded in 10 cm tissue culture dish, grew to reach 80% confluence and then were treated with CdCl₂ (20 μM) for 6 h. After treatment, cells were rinsed twice with ice-cold PBS and lysed with Nonidet P-40 lysis buffer (Sigma). SureBeads (100 μL) (BioRad, Hercules, CA, USA) were transferred into 1.5 mL Eppendorf tube. PBST (PBS plus 0.1% Tween 20) washed three times and discarded supernatant by magnetic separator. Then 200 μL IP antibodies were adhered to SureBeads in shaker for 20 min. Cell lysates were subjected to IP with mouse anti-RIPK3 or anti-RB beads at room temperature for 1 h. Then, the beads were washed three times in PBST. The proteins were eluted by 1× SDS buffer and analyzed by Western blot. Mitochondrial fraction was extracted from fresh mouse liver tissues using the same procedure as for cells. Isolated mitochondrial fraction was used for Co-IP experiment with the same method described as above.

Zhang S., Che L., He C., Huang J., Guo N., Shi J., Lin Y, & Lin Z. (2019). Drp1 and RB interaction to mediate mitochondria-dependent necroptosis induced by cadmium in hepatocytes. Cell Death & Disease, 10(7), 523.

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Western Blot Analysis of Protein Samples

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Isolated islets were sonicated and extracted in a Nonidet-P40 lysis buffer (Sigma-Aldrich; St Louis, MO, USA). Proteins were separated via electrophoresis on either a 5%, 7.5%, or 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). Primary antibodies used for detection are listed in Supplementary Table 2. Proteins were detected using ECL™-Plus Western Blot detecting reagents (Perkin-Elmer, Wellesley, MA, USA) and imaged using the Versadoc Imaging System (Bio-Rad Laboratories). Image Lab software (Bio-Rad Laboratories) was used to quantify band intensities using densitometry, and data were normalized to total or appropriate loading controls [17 (link), 30 (link), 31 (link)].

Peart J., Li J., Lee H., Riopel M., Feng Z.C, & Wang R. (2017). Critical role of β1 integrin in postnatal beta-cell function and expansion. Oncotarget, 8(38), 62939-62952.

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Western Blot Analysis of Signaling Proteins

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Cells were lysed in 0.5% Nonidet P-40 lysis buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Following electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane, Immobilon-P (Millipore Co., Milford, MA, USA). Membranes were blocked with Tris-buffered saline-Blotto/Blotto B (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour and subsequently incubated overnight with antibodies directed against α-CaMKII, p-α-CaMKII, p-CREB, CREB, p-c-Jun, c- Fos, Lamin B1, HIF-1α, VEGFR-1, VEGFR-2 or β-actin (Santa Cruz Biotechnology and Cell Signaling Technology, Beverly, MA, USA). Signals were detected using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit (ECL; Amersham Biosciences, Pittsburgh, PA, USA) [14 (link)]. Band density was measured using ImageJ software and normalized to β-actin.

Daft P.G., Yang Y., Napierala D, & Zayzafoon M. (2015). The Growth and Aggressive Behavior of Human Osteosarcoma Is Regulated by a CaMKII-Controlled Autocrine VEGF Signaling Mechanism. PLoS ONE, 10(4), e0121568.

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Western Blot Analysis of Cell Lines

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All the cell types (Bel-7402, SK-HEP-1, SMMC7721, BGC-823, Hep3B, HepG2 and Caco-2) were seeded (1×10⁷ cells/ml) in 100-mm cell culture dishes. Total protein was then extracted from the cells using 1% Nonidet P-40 lysis buffer (Sigma-Aldrich; Merck KGaA). The protein concentration was measured using the Lowry method. The protein lysates (30 µg protein/lane) were subsequently separated using 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (PVDF; EMD Millipore, Billerica, MA, USA). The blocking reagent used was 5% bicinchoninic acid, and the PVDF membranes were blocked at 37°C for 1 h. These membranes were incubated with the primary antibody, as aforementioned (dilution, 1:100), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (dilution, 1:100; catalog no. ZB-2301; Beijing ZSGB-BIO Co. Ltd) at room temperature. Protein expression was visualized using a Super Signal Protein Detection kit (Pierce; Thermo Fisher Scientific, Inc.). The membranes were then stripped and re-probed with anti-PEPT1 (dilution, 1:1,000), and an anti-β-actin primary antibody (dilution, 1:1,000; catalog no. sc-47778; Santa Cruz Biotechnology, Inc.) served as a loading control. Each experiment was repeated three times.

Gong Y., Zhang J., Wu X., Wang T., Zhao J., Yao Z., Zhang Q., Liu X, & Jian X. (2017). Specific expression of proton-coupled oligopeptide transporter 1 in primary hepatocarcinoma-a novel strategy for tumor-targeted therapy. Oncology Letters, 14(4), 4158-4166.

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