Development of a medium throughput whole-cell microtiter plate Thr286 autophosphorylation assay for CaMKIIα using ELISA

Line B. Palmelund, Geeske M. van Woerden, Hans Bräuner-Osborne, Petrine Wellendorph*

*Corresponding author for this work

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Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a multifunctional Ser/Thr kinase involved in several neuronal signaling pathways including synaptic plasticity. CaMKIIα autonomous activity is highly dependent on Thr286 autophosphorylation (pThr286), which is widely used as a readout for its enzymatic activity. To readily characterise compounds and potential drug candidates targeting CaMKIIα, a simple, generic cell-based assay for quantification of pThr286 levels is needed. In this study, we present a cell-based assay using an adapted ELISA as a suitable and higher throughput alternative to Western blotting. In this 96-well plate-based assay, we use whole HEK293T cells recombinantly expressing CaMKIIα and apply a phospho-specific antibody to detect pThr286 levels by chemiluminescence. In parallel, total CaMKIIα expression levels are detected by fluorescence using an Alexa488-conjugated anti-myc antibody targeting a C-terminal myc-tag. By multiplexing chemiluminescence and fluorescence, phosphorylation levels are normalised to CaMKIIα total expression within each well. The specificity of the assay was confirmed using a phosphodead mutant (T286A) of CaMKIIα. By applying Ca2+ or known CaMKIIα inhibitors (KN93, tatCN21 and AS100105) and obtaining concentration-response curves, we demonstrate high sensitivity and validity of the assay. Lastly, we demonstrate the versatility of the assay by determining autophosphorylation levels in CaMKIIα patient-related mutations, known to possess altered pThr286 responses (E109D, E183V and H282R). The established assay for CaMKIIα is a reproducible, easily implemented, and facile ELISA-based assay that allows for reliable quantification of pThr286 levels.

Original languageEnglish
Article number107226
JournalJournal of Pharmacological and Toxicological Methods
Publication statusPublished - 1 Nov 2022

Bibliographical note

Funding Information:
This work was supported by the Independent Research Fund Denmark (Grant number 8020-00156B ), Simon Fougner Hartmann's Family Foundation and the Drug Research Academy .

Publisher Copyright: © 2022 The Authors


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