3rd Annual Academic Symposium - Satish Rekulapally

Calcium/Calmodulin-dependent protein kinaseII (CaMKII) is a multifunctional protein kinase that phosphorylates and regulates activity of many substrates in various tissues. Traditional CaMKII activity assays rely on incorporation of radioactivity onto a CaMKII-substrate by utilizing a γ-P32 ATP or on anti-phospho-CAMKII antibodies and are limited by cost and time. Also, γ-P32 ATP has a short half life and can pose health risks to the researchers. Hence, there is a need for a non-radioactive CaMKII activity assay that is as reliable, reproducible and robust as western blotting or radioactive CaMKII activity assays. The goal of the project is to develop a reliable non-radioactive method for the quantification of CaMKII activity in vitro. Modifications could be made to the traditional CaMKII activity assay in order to make it non-radioactive. The important modification is the use of non-radioactive ATP instead of a radio-labeled ATP. After spotting the reaction mixture (as in traditional kinase assay) on Whatman P81 paper (a cationic exchanger), the peptide autocamtide-2 could be eluted into a NaCl solution (with basic pH) after washing off all the other contents with phosphoric acid. The eluted peptides could be separated by liquid chromatography (LC) and a shift of 80 Da could be detected by mass spectrometry (MS) if phosphorylation of the peptide has occurred.