TUNEL assay kit was obtained from JIANGSU KEYGEN BIOTECH CO.LTD (Nanjing, China). ECL Western blotting detection system was from Vazyme biotech co., ltd (Nanjing, China). The following antibodies were used: anti-p-Ser473-Akt, anti-Akt, anti-PI3Kp110α (Santa, Cruz, CA, USA), anti-β-catenin (BD, San Diego, CA, USA), anti-p-Ser9-GSK3β, anti-GSK3β, anti-caspase 3, anti-cleaved caspase 3, anti-Bcl-2, anti-Bax, anti-β-actin (Bioworld, St. Louis, MN, USA), anti-TH, (Sigma-Aldrich, St. Louis, MO, USA), anti-DAT (Proteintech, Wuhan, China), anti-NeuN(Abcam, Cambridge, UK). Goat anti-rabbit IgG conjugated with horseradish peroxidase and BCA Protein Assay Kit were from Pierce (Rockford, IL, USA). DAB Peroxidase substrate kit was purchased from Vector Laboratories (Burlingame, USA).
Anti caspase 3
Manufactured by Bioworld Technology
Sourced in United States, China
Anti-caspase-3 is a laboratory reagent used in research applications. It is an antibody that specifically binds to and detects the caspase-3 protein, which plays a key role in the process of apoptosis, or programmed cell death. The core function of Anti-caspase-3 is to facilitate the identification and quantification of caspase-3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti bcl 2, by Bioworld Technology (5 mentions)
Anti β actin, by Bioworld Technology (5 mentions)
Anti bax, by Bioworld Technology (4 mentions)
Anti cytochrome c, by Bioworld Technology (3 mentions)
Anti gsk 3β, by Bioworld Technology (2 mentions)
Mouse anti bax, by Santa Cruz Biotechnology (2 mentions)
Mouse anti bcl 2, by Santa Cruz Biotechnology (2 mentions)
Cleaved caspase 3, by Bioworld Technology (2 mentions)
Mouse anti gr, by Santa Cruz Biotechnology (2 mentions)
Anti cox 4, by Bioworld Technology (2 mentions)
Anti gapdh, by Bioworld Technology (2 mentions)
Anti p akt ser473, by Santa Cruz Biotechnology (1 mentions)
Anti th, by Merck Group (1 mentions)
Anti β catenin, by BD (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Anti pi3kp110α, by Santa Cruz Biotechnology (1 mentions)
Anti cleaved caspase 3, by Bioworld Technology (1 mentions)
Anti akt, by Santa Cruz Biotechnology (1 mentions)
Anti neun, by Abcam (1 mentions)
Proteinase inhibitors, by Vazyme (1 mentions)
11 protocols using anti caspase 3
1
TUNEL Assay and Western Blotting Protocols
2
Western Blot Analysis of Cellular Proteins
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The colon mucosa or splenic mononuclear cells, hucMSCs, and RAW 264.7 cells were homogenized and modified in RIPA lysis buffer, added with proteinase inhibitors (Vazyme Biotech, Shanghai, China). Protein samples (two hundred micrograms) were separated on a 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Sources and dilution factors of primary antibodies were the following: anti-caspase-3 (1 : 800; Bioworld), anti-PCNA (1 : 1000; Cell Signaling Technology), anti-15-lox-1 (1 : 1000; Abcam), and anti-β-actin (1 : 800; Bioworld). After incubation with the primary antibodies overnight at 4°C, the blots were incubated with the secondary antibodies for 1 h at room temperature and then were visualized by chemiluminescence (Millipore, USA) and detected by using the imaging software (GE Healthcare, Life Sciences, USA).
3
Serum-starved U87 Cells: Protein Expression
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Serum-starved U87 cells were treated with PBS, IR (10 Gy), rHGFK1 (40 μg), or in combination for 48 h. The cells were then collected, rinsed in ice-cold PBS and lysed in lysis buffer containing protease inhibitor. Denatured protein was separated by 10% SDS polyacrylamide gel and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% BSA in TBS-Tween 20 (0.05%, v/v), the membrane was incubated overnight at 4 °C with the primary antibodies: anti-MET (Cat. 8198), anti-MET-Y1001 (Cat. 3153P), anti-Chk1-S345 (Cat. 2348T), anti-Chk2-T68 (Cat. 2197T), anti-Chk1 (Cat. 9931T), anti-Chk2 (Cat. 2360T), anti-ATM-S1981 (Cat. 13050S), anti-ATM (Cat. BS60023, Bioworld, St Louis Park, MN, USA), anti-Caspase 3 (Cat. 9665S), anti-PARP (Cat. 9542) and anti-β-actin (Cat. sc-47778, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). All antibodies were purchased from Cell Signalling Technology, unless otherwise specified. After washing with TBST, the membrane was incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibody for 1 h at room temperature. Following several washes, peroxidase activity was visualised with an Immobilon Western HRP kit (Millipore).
4
Protein Expression Analysis of Apoptosis Regulators
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Total protein was extracted using RIPA buffer containing protease K inhibit and quantified using a NanoDrop 2000 spectrophotometer. Proteins separated were by using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk and incubated samples at 4°C overnight after additions of anti-EZH2 (1:500 dilution, Bioworld, MN, USA), anti-Bcl-2 (1:200 dilution, Bioworld, MN, USA), anti-Bax (1:500 dilution, Bioworld, MN, USA), anti-Mcl-1 (1:200 dilution, Bioworld, MN, USA), or anti-Caspase3 (1:300 dilution, Bioworld, MN, USA) antibodies. The membranes were then incubated with corresponding secondary antibody (1:2000 dilution, Abcam, Cambridge, MA, USA). Blots were visualized by using chemiluminescence kits (Beyotime Biotechnology, Haimen, China).
5
Icariin Alleviates Corticosterone-Induced Stress
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ICA (the purity is 98.93%) was purchased from Shanghai Ronghe Medical Science Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) was used to prepare ICA stock solutions and diluted with sterile normal saline (DMSO concentration: 0.1%) (35) . Fluoxetine (Flx) was purchased from Eli Lilly and Company (Suzhou, China) and diluted to 10 mg/mL with a sterile saline solution (36) . Mitochondria Isolation Kit for Tissue (No.89801) was bought from Thermo Fisher Scientific Inc. Rat corticosterone (CORT) ELISA kits from ebioscience (San Diego, CA) were purchased from Beyotime Biotech Inc., China. Mouse anti-GR (1:1000), mouse anti-Bcl-2, mouse anti-Bax (1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-β-actin, anti-Cox-IV, anti-caspase-3, cleaved caspase-3, anti-cytochrome C (1:1000, Bioworld Technology Co., Ltd, Nanjing, China). The tunnel was bought from Wuhan Boster Co., Ltd. (China).
6
Hippocampal Protein Profiling
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Extracted the cytoplasm and mitochondria protein from the hippocampus, and quantified the protein concentrations with BCA (Beyotime, China). Loading buffer (0.1 MTris-HCl buffer (pH 6.8) containing 0.2 M DTT, 4% SDS, 20% glycerol and 0.1% bromophenol blue) was used to dissolve 40-60 µg equal volume protein samples. The samples were separated on 10% SDS-PAGE and then electrically transferred to PVDF membrane at 90 V. PVDF membranes were incubated with TBST (containing 5% skimmed milk) for 1 h at 37 °C and with primary antibodies at 4 °C for 24 h. The primary antibodies used were as follows: mouse anti-Bax, mouse anti-GR (1:1000), mouse anti-bcl-2, (1:400, SantaCruz Biotechnology, USA), antiβ-actin, anti-Cox-IV, anti-caspase-3, cleaved caspase-3, anti-cytochrome c (1:1000, Bioworld Technology, China). The blots were thoroughly washed with TBST and incubated at 37 °C with the secondary antibody in TBST containing 5% skimmed milk powder for 1 h. After that, the signal was tested by enhanced chemiluminescence (ECL kit, Millipore, USA). Cox-IV was used as an internal reference for proteins in mitochondria, while β-actin was used in the cytoplasm. The membranes were imaged and analyzed using the Quantity One Image Analysis Software (Syngene, U.K.) (49) .
7
Apoptosis Pathway Protein Analysis
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The activity of caspase-3, CHOP, and Bcl-2 was analyzed by western blotting. Following cells transfection and subsequent treatment with cisplatin or thapsigargin, cells were harvested and resuspended in IP lysis buffer (Beyotime, China), incubated at 4°C for 30 minutes with occasional vortexing. Subsequently, cells were centrifuged at 12,000 rpm for 10 minutes at 4°C. The following western blot assay was performed according to aforementioned protocol. The primary antibodies were anti-caspase-3 (1: 1,000, Bioworld Technology, USA), anti-Bcl-2 (1: 1,000, Abcam, UK), anti-CHOP (1: 1,000, Santa Cruz Biotechnology) and anti-GAPDH (1: 10,000, Bioworld Technology, USA).
8
Western Blot Analysis of Apoptotic Proteins
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Cells were lysed on ice and centrifuged at 15,000 r/min at 4°C for 20 min. Supernatants were collected and protein concentrations were measured by bicinchoninic acid (BCA) protein assay (Beyotime, China). All of the samples were boiled at 100°C for 5 min. Equal amounts of protein were resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to nitrocellulose (NC) membranes. Membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated with primary antibodies (anti-Bcl-2, anti-Bax, anti-caspase-9, anti-cytochrome c, anti-NF-κB, anti-Mcl-1, and anti-caspase-3 (Bioworld Technology, China); anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and anti-P53 (Santa Cruz Biotechnology, USA)) at 4°C overnight. The next day, NC membranes were washed three times with Tris-buffered saline with Tween 20 (TBST) and incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG and anti-mouse IgG; Bioworld Technology) at room temperature for 2 h. NC membranes were then washed three times with TBST and signals detected with SuperSignal ECL (Pierce, USA).
9
Omentin Attenuates LPS-Induced Vascular Injury
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LPS (E. coli LPS serotype 0111:B4), sodium pentobarbital, Evans Blue dye, collagenase and trypsin were purchased from Sigma (St. Louis, MO, USA). rh-omentin protein was purchased from GeneTex (Irvine, CA, USA). The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): anti-Scr, anti-phospho-Scr (Try416), anti-Akt, anti-phospho-Akt (Ser473), anti-eNOS, anti-phospho-eNOS (Ser1177), anti-NF-κB Rel and anti-phospho-NF-κB Rel (Ser536). Anti-VE-cadherin, anti-pan-cadherin and anti-cleaved caspase-3 antibodies were purchased from Abcam (Cambridge, UK). Anti-GAPDH, anti-CD31, anti-caspase-3, anti-GSK-3β and anti-phospho-GSK-3β (Ser9) antibodies were purchased from Bioworld Technology (Nanjing, China). Ad-β-gal and full-length human omentin (Ad-omentin) were constructed by Genechem (Shanghai, China). Ad-β-gal was used as a control.
10
Qingrekasen Granule Anticancer Mechanism
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The Qingrekasen Granule was purchased from Xinjiang Uygur Pharmaceutical Co., Ltd. (NO.1807521, Xinjiang, China). Adriamycin (ADR) was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (No. 2007E1, Shenzhen, China), and Benazepril was purchased from Novartis (No. 2007, Beijing, China). The BCA Protein Assay Kit was obtained from Beyotime Biotech Inc. (P0012, Shanghai, China), and Guangzhou Jet Bio-Filtration Co., Ltd. (Guangzhou, China) provided the Elisa plates (12 well strip ×8). HPLC grade methanol and acetonitrile were purchased from Merck KGaA (Darmstadt, Germany). Anti-PI3K and anti-p-PI3K antibodies were purchased from Cell Signaling Technology Inc. (MA, United States). The Anti-AKT, anti-p-AKT, anti-mTOR, anti-P-mTOR, anti-BCL-2, anti-Caspase-3, and Beta-actin antibodies were purchased from Bioworld Technology Inc. (MN, United States). All other reagents were HPLC grade.
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