Following the treatments, cells were washed twice with cold PBS and lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM tetrasodium phosphate, 40 mM β-glycerophosphate, 2 mM EDTA, 2 mM EGTA, 1% Triton X, 10% glycerol, 50 mM sodium fluoride, 0.2 mM sodium orthovanadate, and protease inhibitors. The cell lysates were transferred to 1.5-mL Eppendorf tubes, homogenized, and centrifuged at 10,000×g for 5 min at 4°C. The supernatant was transferred to a fresh tube, and protein concentration was determined using the Bio-Rad Protein Assay Reagent (Bio-Rad, Hercules, CA). Equal amounts of protein samples were resolved on SDS-polyacrylamide gel electrophoresis and transferred onto Amersham Hybond ECL 0.45 μM nitrocellulose membranes (GE Healthcare Bio-Sciences). The membranes were immunoblotted with actin, AMPK, P-AMPKαThr172, Bcl-2, caspase 3 full length, eNOS, P-eNOS (Cell Signaling, Danvers, MA), AT1R, AT2R, or p53 (Santa Cruz, Dallas, TX) antibodies, followed by secondary antibodies. The signals were visualized using Thermo Scientific Pierce ECL Western Blotting Detection reagents (Thermo Fisher Scientific) at VersaDoc 3000 Gel Imaging System (Bio-Rad, Hercules, CA).
Full length caspase 3
Manufactured by Cell Signaling Technology
Sourced in United States
Full-length caspase-3 is a laboratory product offered by Cell Signaling Technology. It is a recombinant protein that represents the full-length form of the human caspase-3 enzyme. Caspase-3 is a key enzyme involved in the execution phase of cell apoptosis (programmed cell death).
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 9, by Cell Signaling Technology (5 mentions)
Hybond, by Cytiva (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Anti γh2ax antibody, by Merck Group (4 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
P ampkα thr172, by Cell Signaling Technology (2 mentions)
Amersham hybond ecl 0.45 μm nitrocellulose membranes, by GE Healthcare (2 mentions)
Protein assay reagent, by Bio-Rad (2 mentions)
P enos, by Cell Signaling Technology (2 mentions)
Thermo scientific pierce ecl western blotting detection reagents, by Thermo Fisher Scientific (2 mentions)
Versadoc 3000 gel imaging system, by Bio-Rad (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Sqstm1 p62, by Cell Signaling Technology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Phosstop phosphatase inhibitor, by Roche (1 mentions)
Odyssey clx imager, by LI COR (1 mentions)
Mcl 1, by Santa Cruz Biotechnology (1 mentions)
Phospho ctd ser2, by Merck Group (1 mentions)
16 protocols using full length caspase 3
1
Protein Expression and Detection Protocol
2
Protein Lysis and Western Blot Analysis in MOLT4 Cells
3
Protein Expression Analysis via Western Blot
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Following the treatments, cells were washed twice with cold PBS and lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM tetrasodium phosphate, 40 mM β-glycerophosphate, 2 mM ethylenediaminetetraacetate (EDTA), 2 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetate (EGTA), 1% Triton X, 10% glycerol, 50 mM sodium fluoride, 0.2 mM sodium orthovanadate and protease inhibitors. The cell lysates were transferred to 1.5-ml Eppendorf tubes, homogenized and centrifuged at 10,000 × g for 5 min. at 4°C. The supernatant was transferred to a fresh tube, and protein concentration was determined using the Bio-Rad Protein Assay Reagent (Bio-Rad). Equal amounts of protein samples were resolved on SDS-PAGE and transferred onto Amersham Hybond ECL 0.45 μM nitrocellulose membranes (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). The membranes were immunoblotted with actin, AMPK, P-AMPKαThr172, Bcl-2, caspase 3 full length, eNOS, P-eNOS (Cell Signaling, Danvers, MA, USA), AT1R, AT2R or p53 (Santa Cruz, Dallas, TX, USA) antibodies, followed by secondary antibodies. The signals were visualized using Thermo Scientific Pierce ECL Western Blotting Detection reagents (Thermo Fisher Scientific) at VersaDoc 3000 Gel Imaging System (Bio-Rad).
4
Protein Extraction and Western Blot Analysis
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Total protein was collected from freshly dissected rat hearts and cell lysates. The nuclear and cytoplasmic proteins of H9c2 cardiomyoblasts were collected using a nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology, Jiangsu, China). Equal amounts of protein were separated on 10% or 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to PVDF membranes (Millipore, Eschborn, Germany). The membranes were blocked for 2 hrs with 5% non‐fat milk at room temperature, then incubated overnight at 4°C with primary antibodies specifically against BRD7 (Sigma‐Aldrich), CHOP (Novus, Littleton, CO, USA), XBP‐1s (Abcam), cleaved caspase‐3, full length caspase‐3, B‐cell lymphoma/leukaemia‐2 (Bcl‐2), Bcl2‐associated X protein (Bax), β‐actin, GAPDH, histone3, phospho‐ERK1/2, total ERK1/2, phospho‐Akt and total‐AKT (all Cell Signaling Technology). After being washed three times, the membranes were incubated with respective secondary antibody for 90 min. at room temperature. Protein contents were visualized using an enhanced chemiluminescent reagent (Bio‐Rad, Hercules, CA, USA).
5
Western Blot Analysis of Apoptosis Markers
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Example 9
Cells were dissolved into the Laemmli sample buffer on the plate. The whole cell extracts were sonicated, and the proteins in the lysate were resolved using a 4-12% SDS polyacrylamide gel, and the resolved proteins were blotted onto a nitrocellulose membrane. Antibodies specific to H2A.X, cleaved caspase-3, full-length caspase-3, or cleaved caspase-9 were purchased from Cell Signaling Technology (Danvers, Mass.). The anti-γH2A.X antibody was purchased from Millipore. The membrane was blocked with 5% nonfat dry milk and incubated individually with each of these antibodies dissolved in the blocking buffer. After incubation with peroxidase-conjugated secondary antibodies, the protein of interest was detected using an ECL kit purchased from Thermo Fisher Scientific (Waltham, Mass.).
6
Western Blot Analysis of Prostate Cancer Cell Markers
7
Apoptosis Induction and Inhibition Assay
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Annexin V-Fluor 488 was from Thermo Fisher Scientific (Waltham, MA, USA). O-phospho-L-serine (OPS), Phorbol 12-myristate-13-acetate (PMA), and cycloheximide (CHX) were from Sigma Aldrich (St. Louis, MO, USA). The hydroxamate-based ADAM17/ADAM10 inhibitor GW280264 (GW) was purchased from Aeobious (Gloucester, MA, USA). Marimastat and the ADAM10 inhibitor GI254023X (GI) were purchased from Tocris Bioscience (Bristol, UK). The neutralizing ADAM17 antibody D1 (A12) was purchased from AdipoGene Life Sciences (Fuellinsdorf, Switzerland, AG-27B-6000PF-C100). Antibodies against EGFR, AKT, full-length caspase-3, cleaved caspase-3, and actin were from Cell Signaling Technology (Danvers, MA, USA). Anti-mCherry antibodies were from Novus Biologicals/Bio-Techne (Wiesbaden, Germany). Anti-ADAM17 antibodies used for Western blotting were from Merck Millipore (Darmstadt, Germany; AB19027) and from Cell Signaling Technology (3976S). Recombinant human killer TRAIL (kTRAIL), which utilizes a linker peptide to increase cross-linking to form a more stable oligomer, was from ENZO Life Sciences (Lörrach, Germany); pan-caspase inhibitor zVAD-fmk (ZVAD) was from BACHEM (Bubendorf, Switzerland). The live dead dye 7-Aminoactinomycin D (7-AAD) was from BioLegend (San Diego, CA, USA). BAPTA-AM was from MedChemExpress (Monmouth Junction, NJ, USA).
8
Protein Expression Analysis by Western Blot
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Example 9
Cells were dissolved into the Laemmli sample buffer on the plate. The whole cell extracts were sonicated, and the proteins in the lysate were resolved using a 4-12% SDS polyacrylamide gel, and the resolved proteins were blotted onto a nitrocellulose membrane. Antibodies specific to H2A.X, cleaved caspase-3, full-length caspase-3, or cleaved caspase-9 were purchased from Cell Signaling Technology (Danvers, Mass.). The anti-γH2A.X antibody was purchased from Millipore. The membrane was blocked with 5% nonfat dry milk and incubated individually with each of these antibodies dissolved in the blocking buffer. After incubation with peroxidase-conjugated secondary antibodies, the protein of interest was detected using an ECL kit purchased from Thermo Fisher Scientific (Waltham, Mass.).
9
Western Blot Analysis of Apoptosis Markers
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Example 9
Cells were dissolved into the Laemmli sample buffer on the plate. The whole cell extracts were sonicated, and the proteins in the lysate were resolved using a 4-12% SDS polyacrylamide gel, and the resolved proteins were blotted onto a nitrocellulose membrane. Antibodies specific to H2A.X, cleaved caspase-3, full-length caspase-3, or cleaved caspase-9 were purchased from Cell Signaling Technology (Danvers, Mass.). The anti-γH2A.X antibody was purchased from Millipore. The membrane was blocked with 5% nonfat dry milk and incubated individually with each of these antibodies dissolved in the blocking buffer. After incubation with peroxidase-conjugated secondary antibodies, the protein of interest was detected using an ECL kit purchased from Thermo Fisher Scientific (Waltham, Mass.).
10
Protein Expression Analysis by Western Blot
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Total cell lyates (TCLs) were prepared from cells using Triton X-100 lysis buffer [0.5% Triton X-100, 1X PBS, 1X protease inhibitors (Roche, 04693132001)]. 40 μg of TCLs was loaded onto 8–15% SDS-PAGE, transferred to the Hybond-C Extra membranes (Amersham Biosciences), and incubated with antibodies against proteins of interest, including β-tubulin III (1:5000, Cell Signaling Technology), LC3B (1:1000, Cell Signaling Technology), p62/SQSTM1 (1:1000, Cell Signaling Technology), full-length caspase 9 (1:1000, Cell Signaling Technology), cleaved caspase 9 (1:1000, Cell signaling technology), full-length caspase 3 (1:1000, Cell Signaling Technology), cleaved caspase 3 (1:1000, Cell signaling technology), phospho-p53 (Ser15) (1:1000, Cell Signaling Technology), p53 (1:1000, Cell Signaling Technology), REST (1:500, BD), MAOA (1:2000, Santa Cruz) and GAPDH (1:3000, Santa Cruz). The signals were visualized using a Pierce ECL Western Blotting substrate (Thermo Fisher Scientific) and imaged via a ChemiDoc Touch imaging system (Bio-Rad).
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