基于微孔板的电泳荧光蛋白激酶a检测方法的开发：与滤膜结合法和荧光偏振检测法的比较

Development of a microplate-based, electrophoretic fluorescent protein kinase a assay: comparison with filter-binding and fluorescence polarization assay formats.

作者信息

Miick Siobhan M, Jalali Shila, Dwyer Brian P, Havens John, Thomas Donald, Jimenez Manuel A, Simpson Mathew T, Zile Betsy, Huss Karen L, Campbell Robert M

机构信息

Gen-Probe Inc., San Diego, CA, USA.

出版信息

J Biomol Screen. 2005 Jun;10(4):329-38. doi: 10.1177/1087057104272909.

DOI:10.1177/1087057104272909

PMID:15964934

Abstract

A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture PKA assay developed uses a positively charged, lissamine-rhodamine-labeled kemptide peptide substrate for the kinase reaction and Nanogen's ElectroCapture HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture PKA assay was validated with both known PKA inhibitors and library compounds. The pK(iapp) results obtained in the ElectroCapture PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats.

摘要

已开发出一种基于微孔板的丝氨酸/苏氨酸激酶蛋白激酶A（PKA）电泳检测方法。所开发的电捕获PKA检测方法使用带正电荷、丽丝胺罗丹明标记的肯普肽底物进行激酶反应，并使用Nanogen的电捕获HTS工作站和384孔层压膜板从激酶反应混合物中电泳分离带负电荷的磷酸化肽产物。电泳分离后，使用Tecan Ultra384荧光读数仪对罗丹明标记的磷酸肽产物量进行定量。电捕获PKA检测方法已用已知的PKA抑制剂和文库化合物进行了验证。在电捕获PKA检测中获得的pK(iapp)结果与当前放射性滤膜结合检测和基于抗体的竞争性荧光偏振PKA检测形式所产生的结果相当。