Antibodies against caspase 3

Manufactured by Cell Signaling Technology

Sourced in United States

Antibodies against caspase-3 are laboratory reagents used to detect and analyze the expression of the caspase-3 protein. Caspase-3 is a critical effector caspase involved in the execution phase of cell apoptosis (programmed cell death). These antibodies can be used in various techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to study the role of caspase-3 in cellular processes.

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Close spelling variants of this product

Caspase-3 (2138 citations) Antibodies against cleaved caspase-3 (34 citations) Primary antibodies against cleaved caspase-3 (12 citations) Primary antibodies against caspase-3 (8 citations) Antibodies against caspase-3 and caspase-9 (3 citations) Caspases (2 citations) Antibodies against cleaved caspase 3 (#9661) (2 citations) Primary antibodies against caspase-3 and caspase-9 (2 citations) Antibodies against pro-caspase-3 (2 citations) Antibodies against caspase-3 (Cat. no. 9662 (2 citations)

20 protocols using antibodies against caspase 3

Detecting Caspase-3 Expression in INS-1 Cells Treated with GLP-1 Analogues

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Example 19

Cleaved caspase-3 is a key executor in the apoptotic process. In this example, the expression of caspase-3 was detected using western blot analysis.

INS-1 cells treated with GLP-1 analogues were cultured with xx mM glucose and 500 μl. culture medium per well. For the immunoblot analysis, proteins were separated using SDS-PAGE, and then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was probed using antibodies against caspase-3 (Cell Signaling Technology, Danvers, USA), followed by horseradish peroxidase-(HRP) conjugated secondary antibodies (Epitomics, Burlingame, USA). The membrane was visualized using an enhanced chemiluminescence system (GE Healthcare Life Sciences, Buckinghamshire, USA). The levels of protein expression were normalized against β-actin expression.

The results summarized in FIG. 18A show that the expression level of caspase-3 treated with GLP-1 analogues. FIG. 18B shows the degree of reduced expression level of caspase-3 on INS-1 cells upon the incubation with GLP-1 analogues. The results of FIGS. 18A and 18B indicated that some of GLP-1 analogues, including GLP-1-Aib⁸-EG₄-2E-2FA-C16 (Aib⁸-EG₄-C16) and GLP-1-Ala⁸-EG₄-2E-2FA-C16 (Ala⁸-EG₄-C16), can effectively reduce the expression level of caspase-3 in INS-1 cells.

US10993986B2. Pharmaceutical constructs with enhanced binding affinity with albumin (2021-05-04). Immunwork Inc. [TW]. Inventors: Tse-Wen Chang [TW], Hsing-Mao Chu [TW], Chien-Jen Lin [TW].

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Investigating Cell Viability and Apoptosis

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MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-H-tetrazolium bromide, thiazolyl blue tetrazolium bromide), propidium iodide (PI), rhodamine-123 (Rh123), N-acetyl-L-cysteine (NAC), and Hoechst 33342 were purchased from Sigma (St. Louis, MO). TFP, 5-fluorouracil (5-FU) and oxaliplatin were purchased from Innochem (Beijing, China). Z-LE(OMe)HD(OMe)-FMK (#KGA8261) was purchased from Nanjing KeyGen Biotech (Nanjing, China). For all in vitro assays, TFP was dissolved in DMSO as a 20 mM stock solution. It is dissolved in DMSO/Cremophor EL/saline at 2.5:12.5:85 v/v for the in vivo experiments. Antibodies against caspase-3 (#9664s), cyclin-dependent kinase (CDK) 2 (#2546), cyclin D1 (#2978), P27 (#3688), AKT (#4658s), p-AKT (#4060s), NF-κB P65 (#8242), and p-NF-κB P65 (#3033) were purchased from Cell Signaling Technology. Antibodies against Bax (#610982), Bcl-2 (#2610538), cyclin E (#51-14596R), mouse PD-L1 (#558091), and mouse PD-1 (#562671) were purchased from BD Bioscience. Antibodies against β-actin (#200068-8F10), and CDK4 (#200540) were purchased from Zen Bioscience. Antibodies against human PD-L1 (#329707 ), mouse CD45 (#103112), mouse CD4 (#100408), and mouse CD8 (#100706) were purchased from BioLegend. Secondary antibodies were purchased from Zhongshan Jinqiao Biotechnology Group.

Xia Y., Jia C., Xue Q., Jiang J., Xie Y., Wang R., Ran Z., Xu F., Zhang Y, & Ye T. (2019). Antipsychotic Drug Trifluoperazine Suppresses Colorectal Cancer by Inducing G0/G1 Arrest and Apoptosis. Frontiers in Pharmacology, 10, 1029.

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Cell Apoptosis Signaling Protocol

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Phosphate-buffered saline, dimethyl sulfoxide, Dulbecco modified Eagle’s medium, fetal calf serum, L-glutamine, penicillin, and streptomycin were obtained from Sigma-Aldrich (St Louis, MO, USA). Antibodies against caspase-3, Bcl-2, becn-1, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). All other reagents were of the highest purity among commercially available products.

Han A.R., Yang J.W., Na J.M., Choi S.Y, & Cho S.W. (2019). Protective effects of N,4,5-trimethylthiazol-2-amine hydrochloride on hypoxia-induced β-amyloid production in SH-SY5Y cells. BMB Reports, 52(7), 439-444.

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Western Blot Analysis of Protein Samples

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The concentration of protein samples extracted from tissues and cells was determined using the BCA Protein Assay kit (Bio-Rad, Mississauga, ON, Canada). Protein samples were separated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking in 5% nonfat milk for 2 hours at room temperature, the membranes were treated with antibodies against caspase 3, cleaved caspase 3 and IGF-1 (Cell Signaling Technologies) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 48°C, followed by secondary antibodies at room temperature for 1 hour. Western blot bands were quantified using Odyssey v1.2 software by measuring the band intensity (area × OD) for each group. GAPDH served as an internal control.

Wang R., Bao H., Zhang S., Li R., Chen L, & Zhu Y. (2018). miR-186-5p Promotes Apoptosis by Targeting IGF-1 in SH-SY5Y OGD/R Model. International Journal of Biological Sciences, 14(13), 1791-1799.

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Shikonin-induced Apoptosis Signaling

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Shikonin and dimethylsulfoxide were purchased from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The purity of Shikonin was >98%. Antibodies against caspase-3, poly ADP-ribose polymerase (PARP), c-Myc, p-ERK1/2, ERK1/2, p38 MAPK, p-JNK and JNK were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody against p-p38 MAPK was purchased from Merck KGaA. Goat anti-rabbit, goat anti-mouse and β-actin antibodies were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China).

Shan Z.L., Zhong L., Xiao C.L., Gan L.G., Xu T., Song H., Yang R., Li L, & Liu B.Z. (2017). Shikonin suppresses proliferation and induces apoptosis in human leukemia NB4 cells through modulation of MAPKs and c-Myc. Molecular Medicine Reports, 16(3), 3055-3060.

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Investigating Quercetin and Cisplatin Interactions

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Quercetin and cisplatin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Caspase inhibitor z-VAD-fmk was purchased from Bio Mol. Antibodies against caspase-3, caspase-8, caspase-9, xIAP, IκBα, α-tubulin, and GAPDH were purchased from Cell Signaling Technology (Boston, USA). The antibody against cytochrome c was acquired from Epitomics (Burlingame, USA) and the antibody against PARP was purchased from BD Company (Franklin, USA). Goat anti-mouse IgG, FICT conjugated, was obtained from CW BIO (Beijing, China). Hoechst33342 was purchased from GenMed (Boston, USA). The secondary antibodies (horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG) and the enhanced chemiluminescence substrate were purchased from BIO-RAD (Berkeley, USA). All other common chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Li X., Guo S., Xiong X.K., Peng B.Y., Huang J.M., Chen M.F., Wang F.Y, & Wang J.N. (2019). Combination of quercetin and cisplatin enhances apoptosis in OSCC cells by downregulating xIAP through the NF-κB pathway. Journal of Cancer, 10(19), 4509-4521.

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Subcellular Fractionation and Western Blot Analysis

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Nuclear/cytoplasmic fractionation was carried out using the Cell Fractionation kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer's instructions, and the whole cell lysates were extracted using RIPA Buffer (Cell Signaling Technology). Western blot analysis was performed according to a standard method, as previously described (30 (link)). Antibodies against YAP (Cat. no. ab81183), TAZ (Cat. no. ab93362), MST1 (Cat. no ab76822), SAV1 (Cat. no. ab105105), Bcl-2 (Cat. no. ab194583) and Bcl-xL (Cat. no. ab32370) were purchased from Abcam (Cambridge, MA, USA), and antibodies against caspase-3 (Cat. no. 9662) and caspase-9 (Cat. no. 9501) were from Cell Signaling Technology. The membranes were stripped and reprobed with an anti-α-tubulin antibody (Sigma-Aldrich) as the loading control.

Xu M., Xiao J., Chen M., Yuan L., Li J., Shen H, & Yao S. (2018). miR-149-5p promotes chemotherapeutic resistance in ovarian cancer via the inactivation of the Hippo signaling pathway. International Journal of Oncology, 52(3), 815-827.

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Western Blotting for Protein Analysis

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Western blotting was performed according to standard methods. Cell lysates were extracted using NP-40 lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% NaDC, 0.1% SDS), and protein concentrations in the cell lysates were measured using the BCA Protein Assay kit (Thermo Scientific). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. After incubation with PBS with 0.2% Tween 20 (PBST) containing 5% skimmed milk powder (SMP) for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies diluted at 1:500–1:5000 in PBST containing 5% SMP. After washing five times with PBST, the membranes were incubated with secondary antibodies diluted at 1:10,000 in PBST containing 5% SMP at room temperature for 1 h. After washing five times again with PBST, the bound antibodies were visualized using ECL chemiluminescence (Thermo Scientific) on X-ray films (Carestream Health, Shanghai, China). Actin was used as a loading control. Antibodies against GABPB1 (1:1000), PRDX5 (1:1000), actin (1:5000), eIF4A (1:1000), Flag (1:2000) and horseradish peroxidase-conjugated secondary antibodies were purchased from ProteinTech (Chicago, IL, USA). Antibodies against caspase-3 (1:1000) and LC3 I/II (1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Qi W., Li Z., Xia L., Dai J., Zhang Q., Wu C, & Xu S. (2019). LncRNA GABPB1-AS1 and GABPB1 regulate oxidative stress during erastin-induced ferroptosis in HepG2 hepatocellular carcinoma cells. Scientific Reports, 9, 16185.

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HIV Protein Assay Using Recombinant Antigens

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Recombinant HIV IIIB Oligomeric Glycoprotein gp120 (Baculovirus)(product#1061), and Recombinant tat HIV-1IIIB (product#1002) were obtained from ImmunoDX (USA). Primary antibody against mouse TREM-1(MAB1187) was purchased from R&D systems (USA). Antibodies against caspase3, cleaved caspase3, PARP, cleaved PARP and Bcl-2 were obtained from Cell Signaling Technology (USA), and all other antibodies were purchased from Santa Cruz Biotechnology (USA).

Yuan Z., Fan X., Staitieh B., Bedi C., Spearman P., Guidot D.M, & Sadikot R.T. (2017). HIV-related proteins prolong macrophage survival through induction of Triggering receptor expressed on myeloid cells-1. Scientific Reports, 7, 42028.

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Cell Cycle and Apoptosis Assay Protocol

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Aila was purchased from BioBioPha Co., Ltd (Yunnan, China). Cell cycle kit was obtained from Biotime (Beijing, China). Annexin V-FITC apoptosis kit and bromodeoxyuridine (BrdU) flow cytometry kit were supplied from BD Biosciences (San Jose, CA, USA). RNA extraction kit, PrimeScript RT MasterMix kit and SYBR Premix Ex Taq kit were provided by Takara (Dalian, China). Antibodies against caspase-3, PARP (poly ADP-ribose polymerase) and actin as well as secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against RPA1 and proliferating cell nuclear antigen (PCNA) were obtained from Santa Cruz (CA, USA). Mouse anti-human γ-H2AX monoclonal antibody and goat anti-mouse IgG Alexa Fluro 488 were purchased from Abcam (Cambridge, MA, USA). ECL western blotting substrate kit was supplied from Merck Millipore (Billerica, MA, USA). Amaxa cell line nucleofector kit T was purchased from Lonza (Cologne, Germany). MTT assay kit, cisplatin (DDP) and camptothecin (CPT) were provided by Sigma-Aldrich (St Louis, MO, USA).

Ni Z., Yao C., Zhu X., Gong C., Xu Z., Wang L., Li S., Zou C, & Zhu S. (2017). Ailanthone inhibits non-small cell lung cancer cell growth through repressing DNA replication via downregulating RPA1. British Journal of Cancer, 117(11), 1621-1630.

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