Aβ25-35 was purchased from Sigma-Aldrich (A4559, St Louis, MO, USA). Donepezil Hydrochloride Tablet (Don) was purchased from Eisai, Shanghai, China. HPTQC (Z20080006) was provided by the First Affiliated Hospital of Anhui University of Chinese Medicine. Annexin V-PI kit (Lot.600125a) was purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Protein lysate (Lot. 70126373), PMSF (Lot. 329-98-6), Tween-20 (Lot. 20CS180) and Tris (Lot. 1130K07) were purchased from Beijing Solarbio Science & Technology Co., Ltd. The following antibodies, Caspase3 (Lot. 89Z0306), Caspase9 (Lot. 74z1125), Caspase12 (Lot. 76r9498), CHOP (Lot. 37s3359), and GRP78 (Lot.64f2874), were from Affinity Biosciences (Cincinnati, OH, USA). PVDF membrane (Lot. RB88250) was purchased from Millipore. TRIzol kit (Lot. 15596-026) was purchased from Ambion (Carlsbad, CA, USA). FBS (Lot.10099140C) was purchased from Bio-Rad Laboratories.
Caspase 3
Manufactured by Affinity Biosciences
Sourced in China, United States
Caspase-3 is a member of the cysteine-aspartic acid protease (caspase) family. It is a core executioner of apoptosis, playing a central role in the execution-phase of cell apoptosis. Caspase-3 cleaves and activates other caspases, as well as relevant targets in the cell.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Affinity Biosciences (11 mentions)
Bcl2 associated x (bax), by Affinity Biosciences (8 mentions)
β actin, by Affinity Biosciences (5 mentions)
Cleaved caspase 3, by Affinity Biosciences (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Caspase 9, by Affinity Biosciences (2 mentions)
Grp78, by Affinity Biosciences (2 mentions)
Caspase12, by Affinity Biosciences (2 mentions)
Total protein extraction kit, by Beyotime (2 mentions)
Gsdmd, by Proteintech (2 mentions)
Il 1β, by Proteintech (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Bcl2 associated x (bax), by Proteintech (2 mentions)
P erk, by Affinity Biosciences (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Protein lysate, by Solarbio (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
Aβ25 35, by Merck Group (1 mentions)
Tween 20, by Solarbio (1 mentions)
Fetal bovine serum (fbs), by Bio-Rad (1 mentions)
19 protocols using caspase 3
1
Aβ25-35-Induced Apoptosis Pathway Analysis
2
Protein Expression Analysis in Thyroid Cancer
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The proteins in BCPAP and 8305C cells were extracted using the Total Protein Extraction Kit (Beyotime Biotechnology). The extracted protein (40 μg) were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking in 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against AKT, p-AKT, Wee1, p-Wee1, Cyclin B and β-actin (1:1000; Cell Signaling Technology), CDK1 (1:1000; Abcam), p -CDK1 (1:1000; R&D), Cyclin A (1:1000; Affinity), caspase 3 (1:250; Affinity) and Cleaved caspase 3 (1:250; Affinity). After incubation with secondary antibodies (1:5000 dilution; CST) for 1 h, TBST was used to wash the protein bands, which were visualized using an enhanced chemiluminescence (ECL) detection reagent (Haigene) and detected using the Tanon imaging system (Tanon).
3
Western Blot Analysis of Cardiac Tissue
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The procedure and protocol for Western blot analysis followed that of our recent report (Chen et al. 2022 (link)). In the same way, proteins of the cardiac tissues were lysed by the RIPA lysis buffer, and the protein concentrations were measured with the bicinchoninic acid assay kit (BCA, Servicebio, Wuhan, China). Protein samples were electrophoresed on SDS-PAGE and transferred to the polyvinylidene fluoride (PVDF) membranes (Servicebio, Wuhan, China), which were blocked with 5% nonfat milk for 2 h. The membranes were subsequently incubated with the following primary antibodies against Nrf2 (1:2000, ImmunoWay, TX, USA), Bax (1:1000, Servicebio, Wuhan, China), HO-1 (1:1000, Affinity Biosciences, Jiangsu, China), Bcl-2 (1:1000, Ibid.), caspase-3 (1:1000, Ibid.), and β-actin (1:5000, Ibid.) at 4 °C overnight. Subsequently, the membranes were then incubated with horseradish peroxidase (HRP)-labeled secondary antibody solution at room temperature for 1 h. The proteins were visualized by enhanced chemiluminescent (ECL, Biosharp, Hefei, China) and detected by ChemiScope 6100 imaging system (Clinx, Shanghai, China).
4
Protein Expression Analysis in Heart Tissue
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Heart tissues were lysed with RIPA Lysis Buffer (Servicebio, Wuhan, China), and total proteins were extracted and denatured. The protein concentration was determined using an Ultra-micro spectrophotometer (METTLER TOLEDO, Shanghai, China). Protein (50 µg) was sampled from each group, separated by 10% SDS‑PAGE at constant pressure, and transferred onto PVDF membranes with constant flow (Transfer Membranes, Shanghai, China). Blots were sealed with 5% skim milk on a shaker at room temperature for 1.5 h and then incubated with specific primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cat.#: AF7021), PI3K (Cat.#: AF6241), p-PI3K (Cat.#: AF3241), Akt (Cat.#: AF6261), p-Akt (Cat.#: AF0016), B-cell lymphoma-2 (Bcl-2, Cat.#: AF6139), Bcl-2-associated X (Bax, Cat.#: AF0120), cysteinyl aspartate-specific proteases-3 (caspase-3, Cat.#: AF6311), and cleaved-caspase-3 (Cat.#: AF7022) (Affinity Biosciences, Jiangsu, China) at 1:2000 and 4°C overnight. The specific protein was incubated with a secondary antibody (Cat.#: S0001, Affinity Biosciences, Jiangsu, China) for 50 min at room temperature after washing the PVDF membranes three times with TBST. Proteins were detected using an Intelligent Image Workstation (6000Plus, Guangzhou Biolight Biotechnology Co., Ltd, Guangzhou, China).
5
Rat Model of Pancreatic Injury
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Sprague Dawley (SD) rats, weighing 200–250 g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (animal license No.: SCXK (Chuan) 2020-030). The multifunctional animal impact equipment has been authorized patent (self-developed, patent number: ZL 2016 1 0347341.5). hUC-MSCs were provided by the Chengdu KangErmei Biological Cell Preparation Center (Number: G01210001). The ELISA kits for serum amylase, lipase, and rat IL-6, IL-10, TGF-β, and TNF-α were purchased from Shanghai Jiaocai Biological Technology Co., Ltd. Bax, Bcl-2, and caspase-3, and β-tubulin antibodies were purchased from Affinity Biosciences (Cincinnati, USA). TUNEL kits was purchased from Roche Group (Switzerland).
6
Western Blot Analysis of Inflammation and Apoptosis Markers
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After protein quantification with a bicinchoninic acid (BCA) protein assay kit, samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes, which were incubated overnight at 4°C with the following primary antibodies: ZO-1 (Abcam, MA, USA), occludin (Abcam, MA, USA), caspase-1 (Abcam, MA, USA), ASC (Abcam, MA, USA), NLRP3 (Abcam, MA, USA), IL-1β (Proteintech, Wuhan, China), GSDMD (Proteintech, Wuhan, China), caspase-3 (Affinity Biosciences, OH, USA), Bax (Affinity Biosciences, OH, USA), Bcl-2 (Affinity Biosciences, OH, USA), and β-actin (Affinity Biosciences, OH, USA). Protein bands were detected on a ChemiDoc-It system (Tanon, Shanghai, China) using an ECL kit (Bio-Rad, CA, USA). Protein levels were determined by the use of ImageJ. The density of each band was normalized to its respective loading control (β-actin). Each test was performed in three experiments with different sample batches.
7
Western Blot Analysis of Apoptosis Markers
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The lung tissues were ground under liquid nitrogen, transferred to RIPA buffer (Beyotime Biotechnology Co. Ltd., Shanghai, China) for lysis, and centrifuged to collect the supernatant. The total protein concentration was determined using the Detergent Compatible Bradford Protein Assay Kit (Beyotime Biotechnology Co. Ltd., Shanghai, China). The protein concentration of the samples was adjusted for consistency. A loading buffer was then added and boiling was done to denature the protein. Samples containing 20 μg protein were separated using 10% or 12% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with tris-buffered saline (TBS) containing 5% non-fat milk for 1 h. The PVDF membranes were then incubated with primary antibodies against NLRP3, ASC, Caspase-1, cleaved-Caspase-1, BAX, Bcl-2, Caspase-3, or α-tubulin (Affinity Biosciences LTD, USA) overnight at 4°C. After washing three times with a TBS Buffer containing 0.1% Twain 20, the membranes were incubated with a secondary antibody (HRP-conjugated) for 1 h at room temperature. The signals of ECL chemiluminescence intensity were captured using the FluorChem FC3 system (Protein Simple, USA). The optical density of the protein bands was analyzed using ImageJ software (NIH, Bethesda, USA).
8
Neuroprotective Effects of Edaravone
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Edaravone injection was purchased from Sinopharm Group Guorui Pharmaceutical Co. LTD (China). Sul-F (> 95% pure) was bought from Shijiazhuang Hairui Pharmaceutical Technology Co. LTD (China). 2,3,5-triphenylte-trazolium chloride (TTC) was acquired from Sigma (USA). Hematoxylin-eosin staining (HE) kit and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) kit were purchased from Biyuntian Biotechnology Co. LTD (China). Bcl-2, Caspase3, Bax, CHOP, p-PERK, p-eIF2α, p-IRE1, Caspase12 and ATF4 primary antibodies were purchased from Affinity (China).
9
Protein Expression Analysis of Colon Tissues
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The colon tissues were homogenized with the ice−cold RIPA buffer containing protease inhibitors (Beyotime Biotechnology, Shanghai, China). The colon proteins were collected after centrifugation at 14,000× g for 5 min and determined using BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were separated by 4–12% SDS−PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking at room temperature with a commercial blocking buffer (Beyotime Biotechnology, Shanghai, China), membranes were incubated overnight at 4 °C with the following primary antibodies: Caspase 3, B cell lymphoma2 (Bcl2), Bcl2 associated X (Bax), cleaved Caspase 3 P17, cleaved Caspase 1 P20, and cleaved IL−1β, which were purchased from Affinity Biosciences (Cincinnati, OH, USA); Occludin, Claudin 1, ZO−1, ACS, NLPR3, Caspase 1, IL−1β, GSDMD, and β−actin, which were purchased from Proteintech (Wuhan, Hubei, China). Then, membranes were incubated with HRP−conjugated secondary antibody for 2–2.5 h at room temperature. Finally, the protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, Wilmington, DE, USA) and detected by the ChemiDoc™ imaging system (BIO−RAD, Hercules, CA, USA). The results were quantified using Image J software and normalized to β−actin.
10
Protein Isolation and Western Blot Analysis
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Total protein was isolated using RIPA buffer (Solarbio) supplemented with protease inhibitor cocktails (Sigma). The proteins were separated by SDS-polyacrylamide gel, transferred onto polyvinylidene fluoride membranes (Millipore). After blocking with 5% fat-free milk for 2 h at RT, then membranes were incubated with primary antibodies overnight at 4 °C, followed by peroxidase-conjugated secondary antibodies (1:5000 dilution) for 2 h at RT. The immune complex was visualized by an enhanced chemiluminescence kit (Millipore). The primary antibodies were used at the following dilutions: AKAP8L (1:1000, NBP2-47440; Novus), Lgr5 (1:1000, NBP1-28904; Novus), Oct4 (1:1000, NB100-2379; Novus), CD133 (1:1000, #64326; CST), CD44 (1:1000, #37259; CST), Sox2 (1:1000, #3579; CST), SCD1 (1:1000, DF13253; Affinity), IGF2BP1(1:1000, ab184305; abcam), Cleaved-PARP (1:1000, AF7023; Affinity), PARP (1:1000, #9532; CST), Cleaved-Caspase3(1:1000, #9664; CST), Caspase3(1:1000, #AF6311; Affinity), β-actin (1:2000, #4970; CST), GAPDH(1:1000, #2118; CST).
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