Gsk3β antibody

Manufactured by Santa Cruz Biotechnology

Sourced in United States

The GSK3β antibody is a laboratory research tool used to detect and measure the expression of the GSK3β protein in biological samples. GSK3β is a serine/threonine protein kinase that plays a crucial role in various cellular processes, including metabolism, cell differentiation, and cell survival. The antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to study the expression and localization of GSK3β in different experimental models.

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3 protocols using gsk3β antibody

Stem Cell Differentiation Assay

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The following drugs and reagents were used: lithium chloride (Alfa Aesar B21573, Haverhill, MA, USA), β-catenin antibody (Santa Cruz C2206, Dallas, TX, USA), GSK-3β antibody (Santa Cruz AB15328), Nestin antibody (Abcam ab6142, Cambridgeshire, UK), MAP2 antibody (Merck Millipore AB5622, Burlington, MA, USA), GFAP antibody (Merck Millipore MAB3402C3), neurofilament antibody (Merck Millipore AB15328, MA), DMEM/F12 (Thermo Fisher, Waltham, MA, USA), basic fibroblast growth factor (bFGF, PeproTech, Rocky Hill, NJ, USA), epidermal growth factor (EGF, PeproTech), 0.25% trypsin (Sigma Aldrich, St. Louis, MO, USA), 0.01 mol/L RNase (Sigma Aldrich), 0.5 mg/L propidium iodide staining solution (Boster Biology, Wuhan, China), 5% Chloral hydrate (China), and 4% paraformaldehyde (Boster Biology). The equipment included a cold centrifuge (Sigma Aldrich, USA), an inverted light microscope (Olympus IX70, Tokyo, Japan), a microplate reader (Thermo Fisher), an incubator that was set at a temperature of 37°C and regulated with 5% CO₂ (Thermo Fisher), and flow cytometer (FACSCalibur 2, Becton Dickinson, Franklin Lakes, NJ, USA).

Zhang J., He L., Yang Z., Li L, & Cai W. (2018). Lithium chloride promotes proliferation of neural stem cells in vitro, possibly by triggering the Wnt signaling pathway. Animal Cells and Systems, 23(1), 32-41.

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Western Blot Analysis of GSK3β and β-Catenin

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Western blot analysis was performed, according to the previous method (Cho et al., 2018 (link); Wu et al., 2015 (link)). The right hemisphere was homogenized on ice, and lysed in a lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM EGTA, 1.5 mM MgCl₂·6H₂O, 1 mM sodium orthovanadate, and 100 mM sodium fluoride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Protein samples (30 μg) were separated on sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane. Mouse GSK3β antibody, mouse phosphorylated GSK3β (p-GSK3β) antibody, mouse β-catenin antibody, and mouse phosphorylated β-catenin (p-β-catenin) antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibody (1:2,000; Vector Laboratories) for GSK3β, p-GSK3β, β-catenin, and p-β-catenin were used as secondary antibodies. Using a cold pack and prechilled buffer, membrane transfer was conducted at 4°C. Enhanced chemiluminescence detection kit (Santa Cruz Biotechnology) was used for band detection.

Wang L.R., Kim S.H, & Baek S.S. (2019). Effects of treadmill exercise on the anxiety-like behavior through modulation of GSK3β/β-catenin signaling in the maternal separation rat pup. Journal of Exercise Rehabilitation, 15(2), 206-212.

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Protein Extraction and Western Blot

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Total proteins were extracted from corresponding cells with the radioimmunoprecipitation assay buffer (Thermo Scientific, Rockford, IL, USA) in the presence of Protease Inhibitor Cocktail (Thermo Scientific). Equivalent amounts of protein were resolved and mixed with 5×Lane Marker Reducing Sample Buffer (Thermo Scientific, Rockford, IL, USA), electrophoresed in a 10% sodium dodecyl sulfate-acrylamide gel, and transferred onto Immobilon-P Transfer Membrane (Merck Millipore, Schwalbach, Germany). The signal was detected with an enhanced chemiluminescence detection system (Merck Millipore). The GSK3β antibody was obtained from Santa Cruz Biotechnology (Dallas, Texas, USA), and β-actin antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody was also purchased from Thermo Scientific.

Huang Y., Tan D., Xiao J., Li Q., Zhang X, & Luo Z. (2018). miR-150 contributes to the radioresistance in nasopharyngeal carcinoma cells by targeting glycogen synthase kinase-3β. Journal of cancer research and therapeutics, 14(1).

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