دورية أكاديمية
Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody.
| العنوان: | Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody. |
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| المؤلفون: | Turek TC; High Throughput Screening, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033-1300, USA. tammy.turek@spcorp.com, Small EC, Bryant RW, Hill WA |
| المصدر: | Analytical biochemistry [Anal Biochem] 2001 Dec 01; Vol. 299 (1), pp. 45-53. |
| نوع المنشور: | Comparative Study; Journal Article; Validation Study |
| اللغة: | English |
| بيانات الدورية: | Publisher: Elsevier Country of Publication: United States NLM ID: 0370535 Publication Model: Print Cited Medium: Print ISSN: 0003-2697 (Print) Linking ISSN: 00032697 NLM ISO Abbreviation: Anal Biochem Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: <2000- > : San Diego, CA : Elsevier Original Publication: Orlando Fl : Academic Press |
| مواضيع طبية MeSH: | Antibodies/*chemistry , Fluorescence Polarization/*methods , Peptides/*chemistry , Proto-Oncogene Proteins/*analysis , Proto-Oncogene Proteins/*antagonists & inhibitors, Antibodies/immunology ; Binding, Competitive ; Fluorescent Dyes/chemistry ; Kinetics ; Phosphorus Radioisotopes/chemistry ; Phosphoserine/immunology ; Protein Serine-Threonine Kinases/analysis ; Protein Serine-Threonine Kinases/antagonists & inhibitors ; Proto-Oncogene Proteins c-akt ; Sensitivity and Specificity ; Staurosporine/metabolism ; Staurosporine/pharmacology |
| مستخلص: | A competitive fluorescence polarization (FP) assay has been developed for the serine/threonine kinase, AKT. The FP assay has been formatted in a 384-well microtiter plate and automated using a pipeting workstation with performance suitable for high-throughput screening. The assay design utilizes a fluorescent phosphorylated peptide complexed to a product-specific anti-phospho-serine antibody. When unlabeled substrate is phosphorylated, by the kinase, the product competes with the fluorescent phosphorylated peptide for the antibody. The fluorescent phosphorylated peptide is then released from the antibody into solution resulting in a loss in polarization signal. Seven fluorescent phosphorylated peptides and 19 antibodies were evaluated for this assay. RARTSpSFAEPGK-Fl peptide and anti-phospho-GSK-3alpha Ser21 antibody gave the best affinity and change in polarization signal. The apparent kinetic constants were calculated for the FP assay and were consistent with reported values. The FP assay was validated with known inhibitors and the results compared to a radioactive Flashplate transfer assay, utilizing [(33)P]ATP and a biotinylated substrate, also developed in our laboratory. The IC(50) values generated were comparable between the two methods suggesting the competitive FP assay and Flashplate assay have similar sensitivities and abilities to identify inhibitors during screening. |
| المشرفين على المادة: | 0 (Antibodies) 0 (Fluorescent Dyes) 0 (Peptides) 0 (Phosphorus Radioisotopes) 0 (Proto-Oncogene Proteins) 17885-08-4 (Phosphoserine) EC 2.7.11.1 (Protein Serine-Threonine Kinases) EC 2.7.11.1 (Proto-Oncogene Proteins c-akt) H88EPA0A3N (Staurosporine) |
| تواريخ الأحداث: | Date Created: 20011201 Date Completed: 20020129 Latest Revision: 20211203 |
| رمز التحديث: | 20240627 |
| DOI: | 10.1006/abio.2001.5412 |
| PMID: | 11726183 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 0003-2697 |
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| DOI: | 10.1006/abio.2001.5412 |