Cultured cells were harvested and lysed with NP-40 lysis buffer (Beyotime, Haimen, China) for total protein extraction, whereas xenograft tumor tissues were physically homogenized and lysed with RIPA lysis buffer containing 1% v/v PMSF (Beyotime). Proteins were separated by SDS-PAGE, and transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% milk in TBST buffer (TBS + 0.05% v/v Tween-20), and probed for protein of interest with a specific primary antibody overnight at 4°C, followed by incubation with a secondary antibody for 1 h at room temperature (RT). Anti-YB-1 antibody was purchased from Cell Signaling (Boston, MA, USA), anti-cyclin D, anti-cyclin A, anti-cleaved caspase-3, anti-cleaved PARP, anti-Bcl-2 and anti-Bax antibodies were from Wanleibio (Shenyang, China); HRP-conjugated goat anti-rabbit IgG secondary antibody was purchased from Beyotime. Immune complexes were visualized using the ECL system (Qihai Biotech, Shanghai, China). To verify equal protein loading and transfer, membranes were stripped with stripping buffer (Beyotime) and re-probed with anti-β-actin antibody (Wanleibio).
Anti cleaved caspase 3
Manufactured by Wanlei
Sourced in China
Anti-cleaved caspase-3 is a laboratory reagent used for the detection and quantification of cleaved caspase-3, a well-established marker of apoptosis or programmed cell death. It is a specific antibody that binds to the cleaved form of caspase-3 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti bax, by Wanlei (3 mentions)
Anti bcl 2, by Wanlei (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti caspase 3, by Wanlei (2 mentions)
Np 40 lysis buffer, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Anti cyclin d, by Wanlei (1 mentions)
Anti cleaved parp, by Wanlei (1 mentions)
Stripping buffer, by Beyotime (1 mentions)
Anti β actin antibody, by Wanlei (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti mmp9, by Wanlei (1 mentions)
Anti runx2, by Wanlei (1 mentions)
Anti mmp13, by Wanlei (1 mentions)
Image pro plus version 6, by Media Cybernetics (1 mentions)
Anti grp78, by Cell Signaling Technology (1 mentions)
Anti chop, by Wanlei (1 mentions)
Anti β actin, by Zhongshan Golden Bridge Biotechnology (1 mentions)
6 protocols using anti cleaved caspase 3
1
Comprehensive Protein Expression Analysis
2
Western Blot Analysis of Osteoarthritis Markers
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Western blotting analysis was conducted as previously reported [40 (link)]. Anti-Runx2 (Cat No. WL03358, 1:500, WanleiBio, Shenyang, China), anti-MMP9 (Cat No. WL03096, 1:500, WanleiBio, China), anti-MMP13 (Cat No. WL04694, 1:500, WanleiBio, China), anti-Col10a1 (Cat No. AF6538, 1:500, Beyotime, Shanghai, China), anti-Caspase 3 (Cat No. WL02117, 1:500, WanleiBio, China), anti-Cleaved caspase 3 (Cat No. WL01992, 1:500, WanleiBio, China), anti-β-actin (Cat No. 20536-1-AP, 1:1000, Proteintech, Wuhan, China) primary antibodies were used.
3
Western Blot Analysis of Apoptosis Markers
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Brain tissues were homogenized in the RIPA-DOC buffer supplemented with PMSF and phosphatase inhibitor (Roche). The lysed samples were centrifuged at 12,000 for 30 min at 4°C. The supernatants were used for Western blot analysis. The lysates were resolved on 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membrane at 4°C. The membranes were blocked with 5% skim milk in TBST for 4 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: anti-cleaved caspase-3 (3:5,000, Wanlei), anti-GRP78 (1:1,000; Cell Signaling), anti-CHOP (3:5,000; Wanlei), and anti-β-actin (1:2,000; Zhongshan Golden Bridge Biotechnology). Then, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG secondary antibodies (1:5,000, Zhongshan Golden Bridge Biotechnology) for 1.5 h at room temperature or 3 h at 4°C, followed by chemiluminescence assay. The optical density was quantified by Image-Pro Plus VERSion 6.0 (Media Cybernetics Inc., Bethesda, MD, USA). The experiments were not performed in a blinded manner.
4
Shengqi Fuzheng Injection's Anti-Cancer Potential
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Shengqi fuzheng injection (Z19990065) was provided by livzon Pharmaceutics ltd. (Zhuhai, China). For cell culture, SFI was dissolved in DMEM to different concentration gradients. Gefitinib was purchased from Aladdin Industrial Corporation (Shanghai, China). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Biosharp (Shanghai, China). Dulbecco's modified eagle medium (DMEM), DMSO, Penicillin- Streptomycin Solution, Annexin V-FITC kit were purchased from KeyGen (KeyGen, Nanjing, China). DAPI staining solution, EGF, BCA protein assay kit, Crystal Violet Staining Solution were purchased from Beyotime Biotechnology (Shanghai, China). Anti-p- EGFR(Tyr 1172), anti-EGFR, anti-MEK1/2, anti-p- ERK1/2(thr202/tyr204), anti-ERK1/2, anti-β-actin, goat anti-rabbit IgG H&L (HRP), anti-cleaved-caspase 3, anti-cleaved-caspase 9, anti-Bcl-2, anti-Bax antibodies were purchased from Wanlei Bio. (Shenyang, China). Anti-p-MEK1/2 (Ser217/221) antibody was purchased from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA).
5
Oxidative Stress and Apoptosis Biomarkers
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Diquat (Cat#: 6385-62-2) and melatonin (Cat#: 73-31-4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies anti-Sod1 (Cat#: WL01846), anti-Gpx1 (Cat#: WL02497a), anti-Bax (Cat#: WL01637), anti-Bcl2 (Cat#: WL01556), anti-Cleaved Caspase3 (Cat#: WL01992), anti-P53 (Cat#: WL01919), anti-ZO-1 (Cat#: WL03419), anti-Occludin (Cat#: WL01996), anti-β-catenin (Cat#: WL0962a), and anti-Connexin43 (Cat#: WL02837) were purchased from Wanleibio (Shenyang, China), while anti-Tubulin (Cat#: AF1216) was obtained from Beyotime (Shanghai, China). Unless otherwise indicated, the remaining reagents and chemicals used in this study were purchased from Sigma-Aldrich.
6
Quantitative Protein Analysis in Tumor Cells
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HeLa and CaSki cells and nude mouse tumor tissues were lysed by RIPA lysis buffer containing 1% PMSF and 1% protein phosphatase inhibitor for 30 min and then centrifuged at 14,000 × g at 4°C for 20 min. Collecting the supernatant to store at −80°C. The protein concentration was performed by Bradford assay kit. Cells and tissue lysates were electrophoresed by SDS-PAGE and proteins were transferred to PVDF membrane (Millipore) and then blocked with Tris-buffered saline Tween-20 with 5% non-fat milk for 2 h. Next, they were incubated with primary antibodies diluted with TBST overnight at 4°C, the primary antibodies were as follows: Anti-hnRNP A2/B1 (Bioworld Technology, St. Louis Park, MN, USA; 1:1,000), anti-AKT (Proteintech, Wuhan, China; 1:500), anti-p-AKT (Cell Signaling Technology, Danvers, MA, USA; 1:1,000), anti-p21 (Abcam, Cambridge, UK; 1:2,000), anti-p27 (Abcam; 1:1,000), anti-PI3K (Wanleibio, Shenyang, China; 1:500), anti-caspase-3 (Wanleibio; 1:500), anti-cleaved caspase-3 (Wanleibio; 1:500), β-actin (Bioss, Beijing, China; 1:6,000) was used as a loading control and followed by incubation with secondary antibody (ZSGB-Bio, Beijing, China; 1:90,000) at room temperature for 1 h. Chemiluminent detection was determined by ECL kit. ImageJ software was used to conclude data.
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