Whole cell extracts were obtained from purified mouse B cells or cell lines treated with 1 μM PI3K inhibitors using GST-FISH buffer (10 mM MgCl2, 150 mM NaCl, 1% NP-40, 2% Glycerol, 1 mM EDTA, 25 mM HEPES (pH 7.5)) supplemented with protease inhibitors (Roche), 1 mM phenylmethanesulfonylfluoride (PMSF), 10 mM NaF and 1 mM Na3VO4. Extracts were cleared by centrifugation at 12,000 r.p.m. for 15 min. The supernatants were collected and assayed for protein concentration using the Bio-Rad protein assay method. 20 μg of proteins were loaded on 12% Mini-PROTEIN TGX gels (BIO-RAD), transferred on nitrocellulose membrane (GE Healthcare), blocked with 5% Skim milk (BIO-RAD). Primary antibodies for immunoblotting included: rat monoclonal anti-mouse AID (mAID-2 clone, eBioScience, CAT NO #14-5959-82), mouse monoclonal anti-human AID (ZA001, Life Technologies, CAT NO # 39-2500), rabbit monoclonal anti-PI3K p110δ (Y387, Abcam, CAT NO #32401), rabbit polyclonal anti-β–actin (Sigma, CAT NO #A2066), rabbit monoclonal anti-Phospho-AKT (S473) (D9E, Cell Signaling Technology, CAT NO #4060), rabbit monoclonal anti-AKT (pan) (C67E7, Cell Signaling Technology, CAT NO #4691). Membranes were developed with ECL solution (GE Healthcare). AID protein abundance was measured by ImageJ software and normalized for the β-actin intensity of the corresponding lane.
Za001
Manufactured by Thermo Fisher Scientific
The ZA001 is a laboratory instrument designed for the analysis and measurement of samples. It is a core product in the analytical instrumentation portfolio of Thermo Fisher Scientific, a leading provider of scientific equipment and solutions.
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Lab products found in correlation
2 protocols using za001
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Quantifying AID Protein Expression
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Quantifying AID Protein Expression
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Whole cell extracts were obtained from purified mouse B cells or cell lines treated with 1 μM PI3K inhibitors using GST-FISH buffer (10 mM MgCl2, 150 mM NaCl, 1% NP-40, 2% Glycerol, 1 mM EDTA, 25 mM HEPES (pH 7.5)) supplemented with protease inhibitors (Roche), 1 mM phenylmethanesulfonylfluoride (PMSF), 10 mM NaF and 1 mM Na3VO4. Extracts were cleared by centrifugation at 12,000 r.p.m. for 15 min. The supernatants were collected and assayed for protein concentration using the Bio-Rad protein assay method. 20 μg of proteins were loaded on 12% Mini-PROTEIN TGX gels (BIO-RAD), transferred on nitrocellulose membrane (GE Healthcare), blocked with 5% Skim milk (BIO-RAD). Primary antibodies for immunoblotting included: rat monoclonal anti-mouse AID (mAID-2 clone, eBioScience, CAT NO #14-5959-82), mouse monoclonal anti-human AID (ZA001, Life Technologies, CAT NO # 39-2500), rabbit monoclonal anti-PI3K p110δ (Y387, Abcam, CAT NO #32401), rabbit polyclonal anti-β–actin (Sigma, CAT NO #A2066), rabbit monoclonal anti-Phospho-AKT (S473) (D9E, Cell Signaling Technology, CAT NO #4060), rabbit monoclonal anti-AKT (pan) (C67E7, Cell Signaling Technology, CAT NO #4691). Membranes were developed with ECL solution (GE Healthcare). AID protein abundance was measured by ImageJ software and normalized for the β-actin intensity of the corresponding lane.
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