The total protein from the left ventricle tissue was homogenized using a lysis buffer at a ratio of 100 mg tissue/1 mL buffer. The supernatant was collected, and the protein concentration was measured using the Bradford method (Bio–Rad Laboratories, Hercules, CA, USA). The membranes were incubated overnight at 4 °C with primary antibodies, including Fas Ligand, Fas, FADD, Bax, cytosolic cytochrome c, active caspase-8, active caspase-9, and active caspase-3, IGF-II, p-JNK, SIRT1, PGC-1, Bcl-2, Bcl-xL, and α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were washed and incubated with secondary antibodies, including goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, or goat anti-donkey IgG-HRP (Santa Cruz). The blots were visualized with an enhanced chemiluminescence ECL kit (Millipore Corporation, Billerica, MA, USA). Densitometric analysis was performed using a bioimaging analyzer (LAS-3000; Fujifilm Corporation, Tokyo, Japan).
Active caspase 3
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Active caspase-3 is a laboratory reagent used in the study of cellular apoptosis, or programmed cell death. Caspase-3 is a key executioner enzyme in the apoptotic pathway, and its activation indicates the initiation of this process. This product provides the active, cleaved form of the caspase-3 protein for use in various cellular and biochemical assays.
Automatically generated - may contain errors
Lab products found in correlation
Bcl2 associated x (bax), by Santa Cruz Biotechnology (3 mentions)
Active caspase 9, by Santa Cruz Biotechnology (3 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Las 3000, by Fujifilm (1 mentions)
Cytosolic cytochrome c, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Fas ligand, by Santa Cruz Biotechnology (1 mentions)
Pgc 1, by Santa Cruz Biotechnology (1 mentions)
Sirt1, by Santa Cruz Biotechnology (1 mentions)
P jnk, by Santa Cruz Biotechnology (1 mentions)
Bcl xl, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Roche (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Roche (1 mentions)
6 protocols using active caspase 3
1
Apoptosis Signaling Pathway Analysis
2
Western Blot Analysis of Apoptosis Proteins
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Total protein of cells and tissues was extracted by using RIPA lysis buffer supplemented with protease inhibitors (Roche, Basel, Switzerland). Then, equal amounts of proteins were separated on a 10% SDS-PAGE and transferred to the PVDF membranes (Sigma, St. Louis, USA). After blocking with 5% non-fat milk at room temperature for 1h, the membranes were incubated with Mcl-1 primary antibody (dilution 1:500; Santa Cruz, San Diego, USA), active caspase-3 (dilution 1:1000; Santa Cruz), active caspase-9 (dilution 1:1000; Santa Cruz), Bax (dilution 1:1000; Santa Cruz) and β-actin (dilution 1:2000; Cell Signaling Technology, Danvers, USA) overnight at 4 °C. After washing, the membranes were again incubated with horseradish peroxidase-conjugated secondary antibodies (dilution 1:2500; Cell Signaling Technology). The bands were visualized by ECL Detection kit (HANNOTECH Biosciences, Dongguan, China).
3
Double Immunofluorescent Labeling of Brain Markers
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For double immunofluorescent labeling, sections were prepared as previously mentioned. After air-dried for 1 h, sections were first blocked with solution containing 10 % normal serum (same species as the secondary antibody), 3 % (w/v) BSA, 0.1 % Triton X-100 and 0.05 % Tween-20 for 2 h at room temperature. Then the slides were incubated overnight with primary antibody anti-BTEB2 (rabbit, 1:100; Abcam) and different markers as follows: NeuN (mouse; 1:100; Chemicon), GFAP (mouse; 1:100; Sigma), CD11b (mouse; 1:50; Serotec), active caspase-3 (mouse or rabbit; 1:200; Santa Cruz), at 4 °C. In brief, sections were incubated with both primary antibodies overnight at 4 °C and a mixture of FITC- and TRITC-conjugated secondary antibodies (Jackson ImmunoResearch) for 2 h at 4 °C. In order to detect the morphology of the nucleus, sections were covered with DAPI (0.1 mg/ml in PBS; Sigma) for 1 h at 30 °C. The stained sections were examined with a Leica fluorescence microscope (Leica, DM 5000B; Germany).
4
Western Blot Analysis of Apoptosis Markers
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Total proteins were extracted using radioimmuno precipitation assay (RIPA)-phenylmethylsulfonyl fluoride (PMSF) solution and were quantified by the BCA assay (Beyotime, China). A total of 20 µg of proteins from each sample was subjected to 8% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% skim milk at room temperature for 1 h and incubated overnight with the primary antibodies directly against PARP-1 (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), ERK1/2, phosphorylated ERK1/2 (pERK1/2) (1:500 dilution), active caspase 3 (1:500 dilution), Bcl-2 (1:500 dilution), Bax (1:500 dilution), and β-actin (1:2,000 dilution) overnight at 4°C (all from Santa Cruz Biotechnology). After being washed with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Beyotime) for 1 h at 37°C, and visualized using ECL chemiluminiscence detection reagent.
5
Exploring EGFR-TKI Sensitivity in Cancer
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A drug library containing 381 FDA-approved drugs was purchased from Selleckchem (Houston, TX, United States). Compounds were dissolved in DMSO and stored at −20°C. Drugs diluted with RPMI 1640 medium were used to treat cells at a final concentration of 5 μM. The selected drugs, erlotinib, canertinib and lapatinib, were purchased from Selleckchem. EGFR-TKIs were used in the experiments at a concentration of 0.5–20 μM. The primary antibodies for EGFR, phospho-EGFR(Y1068), HER2, phospho-HER2(Y1248), vimentin, Snail, Slug, cleaved-PARP, and HA-tag were purchased from Cell Signaling Technology (Danvers, MA, United States). E-cadherin, N-cadherin, and active-caspase-3 were purchased from Santa Cruz Biotechnology (Dallas, TX, United States). β-actin was purchased from Sigma-Aldrich (St. Louis, MO, United States).
6
Western Blot Analysis of Apoptosis Markers
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Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher, USA). Protein concentration was measured using Bio-Rad protein assay reagent (Bio-Rad, USA). Equal amounts of proteins (40 μg) of each sample were separated by 10% sodium dodecyl sulfate-polyacrylimide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). PVDF membrane was blocked in skim milk at 25°C for 2 h, and subsequently incubated at 4°C overnight with antibodies against AKT-2, Bcl-2, Bax, active caspase-9, active caspase-3 or GAPDH (all in 1 : 1000 dilution, Santa Cruz, USA) in Tris-buffered saline with Tween-20 (TBST) containing 5% defatted milk. Next, PVDF membrane was incubated with corresponding horseradish peroxidase-conjugated (HRP)-linked secondary IgG antibodies (anti-mouse or anti-rabbit, 1 : 1000 dilution, Santa Cruz, USA) for 1 h at room temperature. The bands of bound secondary antibody were detected with an enhanced chemiluminescence kit (Pierce Biotechnology, USA) and the signals were detected with a SuperSignal Protein Detection kit (Pierce Biotechnology, USA). The band intensity of western blot was quantified subsequent to normalization with the density of GAPDH using ImageJ (National Institutes of Health, USA).
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