Anti caspase 3

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-caspase-3 is a laboratory reagent used to detect and quantify the presence of caspase-3, an enzyme that plays a crucial role in the process of apoptosis or programmed cell death. It is commonly used in research applications to study cellular processes and mechanisms related to apoptosis.

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Lab products found in correlation

Anti nrf2, by Thermo Fisher Scientific (4 mentions) Anti α tubulin, by Merck Group (2 mentions) Anti bim, by GeneTex (2 mentions) Anti bcl 2, by GeneTex (2 mentions) Anti bax, by GeneTex (2 mentions) Anti nf κb p65, by Thermo Fisher Scientific (2 mentions) Anti bax, by Abcam (2 mentions) Anti bcl 2, by Abcam (2 mentions) Anti bcl 2, by Thermo Fisher Scientific (2 mentions) Anti bax, by Thermo Fisher Scientific (2 mentions) Anti e cadherin, by Thermo Fisher Scientific (1 mentions) Anti β actin a2228, by Merck Group (1 mentions) Anti parp, by Thermo Fisher Scientific (1 mentions) Osimertinib, by Selleck Chemicals (1 mentions) Anti fak, by Cell Signaling Technology (1 mentions) Anti paxillin, by Cell Signaling Technology (1 mentions) Mini protean 2, by Bio-Rad (1 mentions) Anti bcl xl, by GeneTex (1 mentions) Anti caspase 7, by GeneTex (1 mentions) Chemiluminescent detection system, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Caspase-3 (46 citations) Anti-cleaved caspase-3 (15 citations) Mouse anti-caspase-3 (2 citations) Alexa Fluor®-488 labeled anti-cleaved caspase-3 (2 citations) Anti-caspase-3 (9H19L2, (2 citations)

11 protocols using anti caspase 3

Osimertinib and FAK Inhibitor Signaling

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Osimertinib was purchased from Selleckchem (S7297), and FAK inhibitor 14 and FAK autophosphorylation inhibitor from Abcam (ab144503). The following antibodies were obtained from Cell Signaling Technology: anti-FAK (#3285), anti-Phos-FAK (Tyr397) (#3283), anti-paxillin (#2542), anti-Phos-paxillin (Tyr118) (#69363), anti-thrombospondin-1 (D7E5F) (#37879), anti-AKT3 (#4059), anti-COL5A1(#37304), anti-E-cadherin (#3195), anti-PARP (#9542), anti-caspase-3 (#9662), anti-cleaved PARP (Asp214) (#5625), anti-N-cadherin (D4R1H) (#13116), anti-E-cadherin (24E10) (#3195), anti-vimentin (D21H3) (#5741), anti-rabbit IgG HRP-linked (#7074P2), and anti-mouse IgG HRP-linked (#7076P2).
The anti-CADM1 (PA3-16744) and anti-BICC (PA5-116342) were purchased from Thermo Fisher. The anti-IGFBP7 (ab74169) and anti-PTPRM (ab231607) were purchased from Abcam. The anti-β-actin (A2228) and anti-RAB32 (HPA025731) were purchased from Sigma-Aldrich.

Kosibaty Z., Brustugun O.T., Zwicky Eide I.J., Tsakonas G., Grundberg O., De Petris L., McGowan M., Hydbring P, & Ekman S. (2022). Ras-Related Protein Rab-32 and Thrombospondin 1 Confer Resistance to the EGFR Tyrosine Kinase Inhibitor Osimertinib by Activating Focal Adhesion Kinase in Non-Small Cell Lung Cancer. Cancers, 14(14), 3430.

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Protein Expression Analysis of Apoptotic Regulators

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Protein extraction was carried out with approximately 2 × 10⁶ cells treated for 6, 16, 24, and 48 h with OXC or Paclitaxel at the corresponding IC₅₀ using a lysis buffer (20 mM Tris HCl pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40, and 10 mM EDTA). The total protein concentration was determined by the Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce), and 25 µg of protein from each sample were subjected to 11% SDS-PAGE polyacrylamide gel electrophoresis using a mini-gel system (mini-Protean II; Bio-Rad Laboratories). Proteins were blotted using polyvinylidene fluoride membranes (PVDF). The transferred membranes were blocked for 1 h with 5% BSA/TTBS (w/v), followed by overnight incubation at 4 °C with the primary antibodies: anti-Bim, anti-Bax, anti-Bcl-2, anti-Bcl-xL, anti-Caspase-7 (GeneTex, Irvine, CA, USA), or anti-Caspase-3 (Thermo Fisher Scientific, Waltham, MA, USA), and anti-α-tubulin (Merck Group, DE, Darmstadt, Germany) was used as a loading control. On the next day, the membranes were incubated with the corresponding secondary antibody, either anti-Mouse IgG or anti-Rabbit IgG (Merck Group, DE, Darmstadt, Germany), for 1 h at room temperature. Bands were visualized on PVDF membranes using the 3,3′-diaminobenzidine tetrahydrochloride (DAB) Substrate Kit detection method (Pierce). ImageJ software was used to semi-quantify and compare band density [5 (link)].

Delgado C., Mendez-Callejas G, & Celis C. (2021). Caryophyllene Oxide, the Active Compound Isolated from Leaves of Hymenaea courbaril L. (Fabaceae) with Antiproliferative and Apoptotic Effects on PC-3 Androgen-Independent Prostate Cancer Cell Line. Molecules, 26(20), 6142.

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Western Blot Analysis of Apoptosis Proteins

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Protein extraction was carried out from approximately 6 million cells treated for 6, 16, 24 and 48 h with the MTAs at the corresponding IC₅₀ using a lysis buffer (Tris HCl pH 8.0 20 mM, NaCl 137 mM, Glycerol 10%, Np40 1%, EDTA 10 mM) [34] , and the protein concentration was determined by Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce). Fifteen μg of protein were subjected to an 11% SDS-PAGE gel electrophoresis using a mini-gel system (Protean II; Bio-Rad Laboratories). Proteins were blotted using polyvinylidene fluoride (PVDF) membranes [35] , [36] . The transferred membranes were blocked in 5% (w/v) BSA/TTBS followed by overnight incubation with the following primary antibodies: anti-α-Tubulin (Sigma–Aldrich); anti-Bim; anti-Bax; anti-Bcl-2 (Gene-Tex); anti-procaspase-3 (Novus) or anti-caspase-3 (Thermo-Fisher Scientific). Next, the membranes were incubated with the correspondent secondary antibody, either anti-mouse IgG (Sigma–Aldrich) or anti-rabbit IgG (Cell Signaling), for 1 h at room temperature. The bands were visualized using a Chemiluminescent Detection System (Thermo-Fisher Scientific) [22] . Image J software was used to quantify and compare the density of bands [37] (link).

Delgado-Carreño C, & Méndez-Callejas G. (2019). Topological properties and in vitro identification of essential nodes of the Paclitaxel and Vincristine interactomes in PC-3 cells. Biomedical Journal, 42(5), 307-316.

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Comprehensive Cardiac Biomarker Evaluation

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The ISO was procured from Sigma (St. Louis, MO, USA). The TAX was purchased from Biosynth Carbosynth (Berkshire, UK). A Cardiac troponin I (cTnI) ELISA kit was obtained from Kamiya (Tukwila, WA, USA). Kits for creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) activities were procured from Spinreact (Girona, Spain). A HO-1 ELISA kit was obtained from MyBioSource (San Diego, CA, USA). The ELISA kits for tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were supplied by R&D Systems (Minneapolis, MN, USA). Anti-NF-κB p65, anti-caspase-3, and anti-Nrf2 antibodies were obtained from ThermoFisher (Waltham, MA, USA; 1:100 dilution), while anti-Bax and anti-Bcl-2 antibodies were supplied by Abcam (Cambridge, MA, USA; 1:100 dilution).

Obeidat H.M., Althunibat O.Y., Alfwuaires M.A., Aladaileh S.H., Algefare A.I., Almuqati A.F., Alasmari F., Aldal’in H.K., Alanezi A.A., Alsuwayt B, & Abukhalil M.H. (2022). Cardioprotective Effect of Taxifolin against Isoproterenol-Induced Cardiac Injury through Decreasing Oxidative Stress, Inflammation, and Cell Death, and Activating Nrf2/HO-1 in Mice. Biomolecules, 12(11), 1546.

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Immunohistochemical Analysis of Oxidative Stress Markers

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For IHC, the deparaffinized and hydrated sections were treated with 0.05 M citrate buffer (pH 6.8) for antigen retrieval followed by 0.3% hydrogen peroxide. The nonspecific antigen-antibody binding was blocked through the addition of normal serum for 20 min. The sections were washed in PBS and probed overnight at 4 °C with anti-NF-κB p65 (ThermoFisher, Waltham, MA, USA), anti-Bax (Abcam, Cambridge, MA, USA), anti-Bcl-2 (Abcam, Cambridge, MA, USA), anti-caspase-3 (ThermoFisher, Waltham, MA, USA), and anti-Nrf2 (ThermoFisher, Waltham, MA, USA). After washing in PBS, anti-mouse secondary antibodies were added to the slides, and DAB was used for color development. Then, the sections were counterstained with Mayer’s hematoxylin, and examined under a light microscope. The staining labelling indices of the caspase-3 and NF-κB p65 were presented as a percentage equivalent field of positive control expression. The immunostaining intensity of anti-Bcl-2 and anti-Nrf2 antibodies was determined through a percent of the positive area using image J analysis software (NIH, Bethesda, MD, USA).

Al-khawalde A.A., Abukhalil M.H., Jghef M.M., Alfwuaires M.A., Alaryani F.S., Aladaileh S.H., Algefare A.I., Karimulla S., Alasmari F., Aldal’in H.K., Alanezi A.A, & Althunibat O.Y. (2022). Punicalagin Protects against the Development of Methotrexate-Induced Hepatotoxicity in Mice via Activating Nrf2 Signaling and Decreasing Oxidative Stress, Inflammation, and Cell Death. International Journal of Molecular Sciences, 23(20), 12334.

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Immunofluorescence analysis of hippocampal neurons

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Primary hippocampal neuronal culture cells seeded in coverslips were fixed in 4% paraformaldehyde for 15 min and washed twice with PBS containing 20 mM glycine, before permeabilization with PBS-glycine containing 0.2% Triton X-100 (15 min). Neurons were treated for 1 h with PBS containing 1% BSA and labelled with the antibodies anti-Nissl (Life technologies, N21480), anti-Ki-67 (Invitrogen, 14-5698-82) and anti-Caspase3 (Thermo Fischer Scientific, 9H19L2). Samples were washed several times and mounted with 30% Mowiol. Samples were observed in a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany). Scale bar: 30 µm. Fluorescence was quantified by Fiji–Image J software (version 2.14.0) considering different fields and dividing the total fluorescence by the number of nuclei in each field.

Raïch I., Lillo J., Rebassa J.B., Capó T., Cordomí A., Reyes-Resina I., Pallàs M, & Navarro G. (2024). Dual Role of NMDAR Containing NR2A and NR2B Subunits in Alzheimer’s Disease. International Journal of Molecular Sciences, 25(9), 4757.

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Exosome Protein Analysis in Parkinson's Disease

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The protein levels were detected as the previously performed [35 (link)]. The exosome samples (exosome from control or PD groups, Exo-control or Exo-PD) were lysed in lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 1 mM PMSF with protease inhibitor mixture, pH 8.0. The supplement was collected after being centrifugated at 14,000 g for 10 min at 4 °C. The loading buffer was added to the protein samples and boiled for 5 min at 98 °C. The protein samples were resolved by 12% SDS-PAGE and then transferred to PVDF membranes for immunoblotting. PVDF membranes were blocked in blocking buffer (1 X TBS containing 5% skim milk) at room temperature for 1 h, followed by diluting with primary antibodies and HRP-conjugated secondary antibodies. The immunoblotting bands were visualized with an ECL kit (Sangon). Images were captured using a chemiluminescence imaging analysis system (Tanon, 5200) and analyzed with the ImageJ.

Antibodies: Anti-ZNF865 (1:1000, PA5-49280, Thermo), Anti-Caspase 3 (1:1000, 700182, Thermo), Anti-CD63 (1:1000,10628D, Thermo), Anti-GAPDH (1:1000, MA1-16757, Thermo), HRP-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (1:5000, Proteintech).

Liu C., Wang Y., Li J.W., Zhu X., Jiang H.S., Zhao H, & Zhang L.M. (2023). MiR-184 Mediated the Expression of ZNF865 in Exosome to Promote Procession in the PD Model. Molecular Neurobiology, 61(6), 3397-3408.

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Histopathological and Molecular Analyses of Liver

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Liver samples fixed in 10% NBF for 24 h were processed for paraffin embedding and 5-µm sections were cut using a microtome. The sections were stained with hematoxylin and eosin (H&E) as previously described [63 ]. To evaluate changes in the expression levels of iNOS, Bax, Bcl-2, caspase-3, and Nrf2, sections from the liver were dewaxed and 0.05 M citrate buffer (pH 6.8) was used for antigen retrieval. The slides were treated with 0.3% hydrogen peroxide (H₂O₂), washed in phosphate-buffered saline (PBS), and probed with anti-iNOS (ThermoFisher Scientific, Waltham, MA, USA; 1:20 dilution), anti-Bax (Abcam, Cambridge, MA, USA; 1:100 dilution), anti-Bcl-2 (Abcam, Cambridge, UK; 1:100 dilution), anti-caspase-3 (ThermoFisher Scientific, Waltham, MA, USA; 1:100 dilution), and anti-Nrf2 (ThermoFisher Scientific, Waltham, MA, USA; 1:100 dilution) overnight at 4 °C. The slides were washed in PBS, incubated with secondary antibodies, and color was developed by incubation with DAB in H₂O₂ [64 ]. Mayer’s hematoxylin was used for counterstaining, and the slides were visualized under a light microscope. The intensity of the developed color was determined in 6 fields/slide using ImageJ (NIH, Bethesda, MD, USA).

Al-Amarat W., Abukhalil M.H., Alruhaimi R.S., Alqhtani H.A., Aldawood N., Alfwuaires M.A., Althunibat O.Y., Aladaileh S.H., Algefare A.I., Alanezi A.A., AbouEl-ezz A.M., Ahmeda A.F, & Mahmoud A.M. (2022). Upregulation of Nrf2/HO-1 Signaling and Attenuation of Oxidative Stress, Inflammation, and Cell Death Mediate the Protective Effect of Apigenin against Cyclophosphamide Hepatotoxicity. Metabolites, 12(7), 648.

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Immunohistochemical Analysis of Apoptosis and Oxidative Stress Markers

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By immersing in xylene and descending graded concentration of ethanol solutions, paraffin-embedded sections were sliced, deparaffinized, and hydrated before being microwave antigen retrieving treated. After that, a 0.3 % hydrogen peroxide per methanol solution was utilized to stop endogenous peroxidase activity. The prepared slides were cooled at ambient temperature, and nonspecific binding was blocked with normal serum for 20 min at ambient temperature. Then, they were blended with anti-Bax, anti-caspase 3, anti-Nrf2, anti-Bcl-2 (all purchased from Invitrogen, Waltham, MA, USA), and anti-NF-κB p65 (purchased from Santa Cruz Biotechnol., Dallas, TX, USA) and preserved 24 hrs in the refrigerator at 4 °C. The 2^ry antibodies were smeared after washing the slides with PBS several times. The color expansion was prompted via incubation with 3,3′-diaminobenzidine-tetrahydrochloride-H₂O₂ solution, and then all prepared sections were stained with Mayer’s hematoxylin and examined by light microscopy. Staining intensity was estimated and achieved as a positive expression% in 1000 cells per 8 HPF for NF-ĸB p65, caspase 3, and Bax, while Nrf2 and Bcl-2 immunostaining was verified via the zone of + ve expression by applying ImageJ evaluation software (NIH, Bethesda, MD, USA).

, & Algefare A.I. (2022). Renoprotective and Oxidative Stress-Modulating Effects of Taxifolin against Cadmium-Induced Nephrotoxicity in Mice. Life, 12(8), 1150.

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Western Blot Analysis of Cell Signaling Proteins

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Samples were solubilized in ice-cold RIPA lysis buffer (Solarbio, Beijing, China) containing a protease inhibitor cocktail (MedChemExpress, Shanghai, China). The protein concentrations were determined using the bicinchoninic acid method (Beyotime, Shanghai, China). Membrane proteins were subjected to SDS-PAGE (Solarbio, Beijing, China) and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, United States) and blocked with 5% non-fat dry milk in tris-buffered saline containing 0.1% tween-20 (TBST) for 1 h before incubation with rabbit anti-CD47 antibody (1:1,000, Abcam), anti-NF-κB (p65; 1:1,000, Cell Signaling, Danvers, MA, United States), anti-p-NF-κB (p-p65; 1:1,000, Cell Signaling), anti-Bcl-2 (1:1,000; Proteintech, Rosemont, IL, United States), anti-GSK-3β (1:1,000, Cell Signaling), anti-p- GSK-3β (Ser9; 1:1,000, Cell Signaling), anti-Bax (1:1,000; Proteintech), anti-caspase-3 (1:2,000; Invitrogen), and mouse anti-GAPDH (1:10,000, Proteintech) in 5% non-fat dry milk/TBST overnight. Then, the membranes were incubated with alkaline phosphatase conjugated Affinipure goat anti-rabbit IgG (H + L; 1:10,000, Proteintech) in TBST for 2 h at 37°C. The membranes were visualized using an ECL detection kit (Pierce Biotech, Rockford, IL, United States). The blots were analyzed and quantified using the ImageJ analysis software.

Xu L.N., Wang S.H., Su X.L., Komal S., Fan H.K., Xia L., Zhang L.R, & Han S.N. (2021). Targeting Glycogen Synthase Kinase 3 Beta Regulates CD47 Expression After Myocardial Infarction in Rats via the NF-κB Signaling Pathway. Frontiers in Pharmacology, 12, 662726.

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