Myricetin (Fig. 1A ) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The compound was dissolved in 100% dimethyl sulfoxide (DMSO). A 100 mmol/L stock solution of Myricetin was prepared and stored as small aliquots at −20°C until needed. We purchased MTT, DMSO, gelatin, and Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit antibodies from Sigma-Aldrich. Growth factor-reduced Matrigel was purchased from BD Biosciences (San Jose, CA, USA). The specific antibodies against PI3K, PDK1, AKT, mTOR, and their phospho forms, anti-procaspase-3 antibody, and the AKT inhibitor LY294002 were purchased from Cell Signaling Technology (Danvers, MA, USA). The HRP-conjugated β-actin, p53, and Bax antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2′,7′-dichlorofluorescein (DCF) diacetate (H2DCFDA) was purchased from Molecular Probes (Invitrogen, Carlsbad, CA, USA).
Anti procaspase 3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-procaspase-3 antibody is a laboratory reagent used to detect and study the expression of procaspase-3, a proenzyme form of the caspase-3 protein. Caspase-3 is a key enzyme involved in the execution phase of apoptosis (programmed cell death). The antibody can be used for applications such as western blotting, immunohistochemistry, and flow cytometry to analyze the presence and distribution of procaspase-3 in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Ly294002, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse and anti rabbit antibodies, by Merck Group (1 mentions)
2 7 dichlorofluorescein dcf diacetate h2dcfda, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Gelatin, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Myricetin, by Merck Group (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Mini protean tgxtm gel, by Bio-Rad (1 mentions)
Anti lin28a antibody, by Cell Signaling Technology (1 mentions)
Anti phospho akt antibody, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
Anti bax antibody, by Cell Signaling Technology (1 mentions)
Anti akt antibody, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Clarity maxtm western ecl substrate, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Image labtm software, by Bio-Rad (1 mentions)
4 protocols using anti procaspase 3 antibody
1
Myricetin Compound Preparation and Analysis
2
Comprehensive Protein Expression Analysis
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The cells or islets were lysed using RIPA buffer (Sigma, USA) with protease inhibitor and phosphatase inhibitor, and the cell lysate was harvested by centrifugation at 13000 rpm for 20 min. The protein samples were separated by Mini-PROTEANⓇ TGXTM Gels (Bio-Rad, USA) and electrophoretically transferred onto membranes. The membranes were incubated with blocking solution containing 5% BSA (Sigma) for 1 h, and further incubated with anti-Lin28a antibody, anti-Bax antibody, anti-cleaved-caspase3 antibody, anti-pro-caspase3 antibody, anti-phospho-Akt antibody, anti-Akt antibody, anti-phospho-mTOR antibody and anti-mTOR antibody (Cell Signaling, USA), anti-Bcl-2 antibody and anti-PDX-1 antibody (Santacruz, USA), or anti-BETA2 antibody (Abcam, UK). Antibodies were detected using a horseradish peroxidase-conjugated secondary antibody (Santacruz) and Clarity MaxTM Western ECL Substrate (Bio-Rad). The membranes were re-blotted with an anti-actin antibody (Santacruz) and immunoblot images were analyzed by ChemiDocTMXRS+ (Bio-Rad), and the intensity of the bands was quantified using Image LabTM Software (Bio-Rad).
3
Examining miR-455-3p Regulation of Cell Signaling
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After 48 h of transfection with miR-455-3p mimics or inhibitors, the cells were lysed in lysis solution and prepared for protein extraction. The protein concentration was measured by a BCA protein assay kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s instructions. Soluble protein (the same concentration per lane) was resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to nitrocellulose membrane. Then the membranes were blocked with 5% non-fat dried milk and incubated with following primary antibodies overnight at 4oC: anti-p27 kinase inhibition protein (KIP) 1 antibody (P2092, Sigma-Aldrich), anti-p21 antibody (SAB4500065; Sigma-Aldrich), anti- B-cell lymphoma (Bcl)-2 antibody (AB1722, Sigma-Aldrich), anti-Bax antibody (B8429, Sigma-Aldrich), anti-pro-caspase 3 antibody (9662, Cell Signaling Technology), and anti-active caspase-3 antibody (ab2302, Abcam, Cambridge, UK). The membranes were then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. GAPDH was used as a loading control. Reactions were visualized with an enhanced chemiluminescence (ECL) system (Santa Cruz, CA).
4
Protein Expression Analysis by Western Blot
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Western blot was carried out to detect the protein levels according to a previous study (19 (link)). After transfection with the miR-142-5p mimic, inhibitor, and the corresponding negative control for 48 h, the cells were collected to extract the total protein. The protein concentration was determined using the BCA Assay Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instruction. After that, the protein samples were subjected to a 10%–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and blotted onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Thereafter, the cells were washed three times with PBS and blocked in 5% defatted milk in Tris-buffered saline with Tween (TBST) for 1 h. The membranes were then incubated with the following primary antibodies overnight at 4°C: anti-FOXO1 antibody (Abcam, Cambridge, UK), anti-FOXO3 antibody (Abcam), anti-Bcl-2 interacting mediator of cell death (Bim) antibody (Cell Signaling Technology Inc., Beverly, MA), anti-procaspase 3 antibody (Cell Signaling Technology), and anti-active caspase 3 antibody (Cell Signaling Technology). After being washed three times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated antibody for 2 h. GAPDH was used as a loading control. The protein bands were visualized with enhanced chemiluminescence (ECL) (Beyotime, P.R. China).
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