Development of a Time-efficient and Cost-effective Luminescence Assay to Measure the Relative Activity of Protein Kinases
Dr. Robert Newman
The objective of this study is to understand the mechanisms of crosstalk between redox- and phosphorylation-dependent signaling. Due to its sensitivity and ability to measure ADP generation directly, the process was applying the commercially available ADP-Glo assay to measure changes in kinase activity following treatment with various concentrations of H2O2. However, the cost and time-intensive nature of the ADP-Glo assay is nonoptimal for undergraduate research due to limited hours students have to run their assays. Therefore, the aim of the current project is to develop an alternative activity assay that is faster and more cost-effective than the ADP-Glo assay while maintaining sensitivity or the ability to directly measure ADP generated. Consequently, there has been the development of an assay based on the nucleotide diphosphate kinase (NDPK)/uracil triphosphate (UTP)-dependent conversion of ADP to ATP. The NDPK/UTP assay, which utilizes two parallel experiments (i.e., water alone treatment compared to the NDPK/UTP treatment), conserves time by using NDPK/UTP to regenerate the ATP consumed in the kinase assays and comparing the difference with the water alone treatment. The difference in the treatments corresponds to the magnitude of activity change by using the ATP present in the water alone treatment as the baseline to measure activity. Side-by-side comparisons between the NDPK/UTP and ADP-Glo assays, using either ERK2 or p38a, show similar changes in kinase activity toward model peptide substrates following H2O2 treatment. Compared to the ADP-Glo assay, NDPK/UTP-based detection reduces the time-to-detection ~3-fold and is also a fraction of the cost of the ADP-Glo assay.
Kumari, Anjali, "Development of a Time-efficient and Cost-effective Luminescence Assay to Measure the Relative Activity of Protein Kinases" (2019). Undergraduate Research and Creative Inquiry Symposia. 60.