Bell Ryan A V, Storey Kenneth B
Institute of Biochemistry and Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6.
MethodsX. 2014 Aug 8;1:96-101. doi: 10.1016/j.mex.2014.07.003. eCollection 2014.
The efficacy of cellular signal transduction is of paramount importance for the proper functioning of a cell and an organism as a whole. Protein kinases are responsible for much of this transmission and thus have been the focal point of extensive research. While there are numerous commercially available protein kinase assays, the Kinase-Glo luminescent kinase assay (Promega) provides an easy-to-use and high throughput platform for determining protein kinase activity. This assay is said to require the use of a microplate spectrophotometer capable of detecting a luminescent signal. This study shows that:•The ChemiGenius Bioimaging system (Syngene), typically used for visualizing chemiluminescence from Western blots, provides an alternative detection system for Kinase-Glo luminescence.•The novel detection system confers an advantage over traditional luminometers, in that it allows visualization of the luminescent wells, which allows for the real-time analysis and correction of experimental errors (i.e. bubble formation).•Determining kinase kinetics using this detection system produced comparable results to previous studies on the same enzyme (i.e. glycogen synthase kinase 3).
细胞信号转导的效能对于细胞乃至整个生物体的正常运作至关重要。蛋白激酶在很大程度上负责这种信号传递,因此一直是广泛研究的焦点。虽然有许多市售的蛋白激酶检测方法,但Kinase-Glo发光激酶检测法(普洛麦格公司)为测定蛋白激酶活性提供了一个易于使用且高通量的平台。据说该检测法需要使用能够检测发光信号的微孔板分光光度计。本研究表明:
•ChemiGenius生物成像系统(Syngene)通常用于可视化蛋白质印迹的化学发光,它为Kinase-Glo发光提供了一种替代检测系统。
•这种新型检测系统相对于传统的发光计具有优势,因为它可以可视化发光孔,从而允许对实验误差(即气泡形成)进行实时分析和校正。
•使用该检测系统测定激酶动力学产生的结果与先前对同一酶(即糖原合酶激酶3)的研究结果相当。