Caspase 3 rabbit mab

Manufactured by Cell Signaling Technology

Sourced in United States

Caspase-3 rabbit mAb is a laboratory reagent used for the detection and quantification of caspase-3, a key enzyme involved in the execution phase of cell apoptosis (programmed cell death). This monoclonal antibody specifically recognizes the caspase-3 protein and can be used in various immunoassay techniques to study cellular processes related to apoptosis.

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5 protocols using caspase 3 rabbit mab

Apoptosis Induction and Mitochondrial Dynamics

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The apoptosis inducer ATP was purchased from Sigma-Aldrich (St. Louis, USA), the P2X1 antagonist NF449 from Tocris Bioscience (Minneapolis, USA), Fluo-4 AM from Dojindo Laboratories (Mashiki-machi, Japan), cell mitochondria isolation kit from Beyotime Biotechnology (Beijing, China) and Lipofectamine® 3000 transfection kit from Thermo Fisher Scientific (Waltham, USA). An isothiocyanate (FITC)-Annexin V/propidium iodide (PI) apoptosis detection kit, TUNEL in situ cell death detection kit and JC-1 mitochondria membrane potential detection kit were purchased from KeyGen (Nanjing, China); protein A-agarose immunoprecipitation reagent and normal rabbit IgG from Santa Cruz Biotechnology (California, USA); HA-tag rabbit mAb, HA-tag mouse mAb, β-actin rabbit mAb, caspase-3 rabbit mAb, caspase-7 rabbit mAb, caspase-9 rabbit mAb and PARP rabbit mAb from Cell Signaling Technology (Boston, USA); Bax rabbit mAb, Bcl2 rabbit mAb, Cyt C rabbit mAb, P2X1 rabbit pAb and cytochrome C oxidase IV (COXIV) rabbit mAb from Abcam (Cambridge, UK); and DDDDK-Tag Mouse mAb from Abclonal (Boston, USA).

Zhou L.J., Chen M., Puthiyakunnon S., He C., Xia J., He C.Y., Deng S.Q, & Peng H.J. (2019). Toxoplasma gondii ROP18 inhibits human glioblastoma cell apoptosis through a mitochondrial pathway by targeting host cell P2X1. Parasites & Vectors, 12, 284.

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Investigating Cell Signaling Pathways

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Cell culture medium was obtained from Invitrogen (Carlsbad, CA) and fetal bovine serum from Hyclone Thermo (Waltham, MA. #SH300070.03). Antibodies for β-catenin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LC-3 antibody (NB100-2220) was purchased from Novus biological (Littleton, CO).
GAPDH rabbit mAb (#2118), caspase-3 rabbit mAb (#9665), p-GSK3β (Ser9) rabbit antibody (#5558), GSK3β rabbit antibody (#9315), p-AKT (Thr308) rabbit antibody (#9275), p-AKT (Ser473) rabbit antibody (#4060), AKT rabbit antibody (#9272), Cleaved caspase 3 rabbit antibody (#9664) and caspase 3 rabbit antibody (#9662) were all obtained from Cell Signaling (Beverly, MA).
nCDase antibody (Ab174) was a kind gift from Dr. Rick Proia (Genetics of Development and Disease, NIDDK, Bethesda, MD) ^{11 (link)}. The enhanced chemiluminescence kit (ECL) was from ThermoScientific (Rockford, IL). C₆ urea-ceramide was provided by the Lipidomics Core facility at MUSC.

Coant N., García-Barros M., Zhang Q., Obeid L.M, & Hannun Y.A. (2018). AKT as a key target for growth promoting functions of neutral ceramidase in colon cancer cells. Oncogene, 37(28), 3852-3863.

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Membrane Protein Extraction and Western Blot Analysis

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Membrane proteins were prepared using a Membrane Protein Extraction Kit (BioVision, Mountain View, CA) according to manufacturer’s instructions. Total proteins were isolated using a RIPA Buffer (Sigma-Aldrich). Twenty microgram membrane proteins or 30µg total proteins were separated by electrophoresis using 4–15% mini-protean TGX precast gel and transferred to polyvinyl difluoride (PVDF) membranes. Membranes were blocked with 5% dry milk in tris-buffered saline (TBS) (all products from Bio-Rad Laboratories, Hercules, CA) and probed overnight at 4 °C with primary-antibodies including rabbit polyclonal K_V1.3 (1:100; Alomone Lab, Israel), anti-iNOS, anti-TNF-α, anti-IL-1β antibody (1:100, Abcam, Cambridge, MA), cleaved caspase-3 antibody, caspase-3 rabbit mAb (1:1000; Cell Signaling Technology, Danvers, MA), and anti-mouse β-actin monoclonal antibody (1:10,000, Sigma-Aldrich). Membranes were washed (4 times, 10 min each) in TBS with 0.2% Tween (TBS-T) and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit, or anti-mouse secondary antibody (1:10,000, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1h at RT. Labeled proteins were visualized by Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL). Band densities of K_V1.3, iNOS, TNF-α, IL-1β were normalized to their p-actin, and cleaved caspase-3 was normalized to caspase-3 in each sample.

Liu J., Xu E., Tu G., Liu H., Luo J, & Xiong H. (2017). Methamphetamine potentiates HIV-1gp120-induced microglial neurotoxic activity by enhancing microglial outward K+ current. Molecular and cellular neurosciences, 82, 167-175.

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Investigating Cell Signaling Pathways

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Coant N., García-Barros M., Zhang Q., Obeid L.M, & Hannun Y.A. (2018). AKT as a key target for growth promoting functions of neutral ceramidase in colon cancer cells. Oncogene, 37(28), 3852-3863.

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Western Blot Analysis of Protein Biomarkers

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The cell lysates were homogenized in mammalian protein extraction buffer (Sigma). The resulting lysates were centrifuged at 12,000 rpm, and 4°C for 15 min, and the protein contents were measured using a protein-assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, 10–15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed, followed by transfer onto nitrocellulose blotting membranes (Amersham, GE Healthcare Life Science, Germany). The antibodies used for immunoblotting are as follows: Bax rabbit mAb (1:1,000; Cell Signaling Technology), Bcl2 (D17C4) rabbit mAb (1:1,000; Cell Signaling Technology), inducible nitric oxide synthase (iNOS; NOS2) (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), AMPKα (D63G4) rabbit mAb (1:1,000; Cell Signaling Technology), Phospho-AMPKα (Thr172) (D4D6D) rabbit mAb (1:1,000; Cell Signaling Technology), caspase-3 rabbit mAb (1:1,000; Cell Signaling Technology), cleaved caspase-3 (Asp175) (5A1E) rabbit mAb (1:1,000; Cell Signaling Technology), PARP (46D11) rabbit mAb (1:1,000; Cell Signaling Technology), and β-actin (13E5) rabbit mAb (1:2,500; Cell Signaling Technology). The protein bands were visualized using an enhanced chemiluminescence method with an ELC kit (Millipore, Billerica, MA, USA) and were quantified using Quantity 1 v.4.6.7 (Bio-Rad).

Kim K., Kwak M.K., Bae G.D., Park E.Y., Baek D.J., Kim C.Y., Jang S.E., Jun H.S, & Oh Y.S. (2021). Allomyrina dichotoma larva extract attenuates free fatty acid-induced lipotoxicity in pancreatic beta cells. Nutrition Research and Practice, 15(3), 294-308.

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