The CycLex® CaM kinase II Assay Kit is designed to measure the activity of CaM kinase II in cells lines or tissue homogenates, and for screening for CaM Kinase inhibitors or activators. The assay is a simple 96-well ELISA that uses a phospho-specific monoclonal antibody to recognise the phosphothreonine residue in “Syntide-2”, which can be efficiently phosphorylated by CaM kinase II.
Fig 1. Q column elution profile of CaM Kinase II from rabbit brain extract, measured using the Cyclex CaM Kinase II assay kit.
Ca2+ / calmodulin-dependent protein kinase (CaM kinase II) is a ubiquitously expressed, multifunctional protein kinase involved in neurotransmitter synthesis and release, neuronal plasticity and gene expression. CaM kinase II is highly concentrated at synapses that use glutamate as the neurotransmitter. CaM kinase II phosphorylates the glutamate receptor and enhances the ion current, which may contribute to mechanisms of synaptic plasticity for learning and memory. CaM kinase II requires calcium-bound calmodulin for activation and for its ability to phosphorylate and alter the function of a variety of substrates.
Principle of the Assay
The CycLex CaM kinase II Assay Kit is a single-site, semi-quantitative immunoassay for CaM kinase II activity. Plates are pre-coated with a newly designed “Syntide-2”, which can be efficiently phosphorylated by CaM kinase II on a microtiter plate. The detector antibody, MS-6E6, specifically detects only the phosphorylated “Syntide-2”.
The CycLex CaM kinase II Assay Kit can be used to determine the presence of CaM kinase II activity in purification column fractions, or to follow the kinetics of a purified CaM kinase II protein as well as screening for CaM kinase II inhibitors or activators.
To perform the test, the sample is diluted in Kinase Buffer, pipetted into the wells and allowed to phosphorylate the bound substrate in the presence of Mg2+ and ATP. The amount of phosphorylated substrate is measured by binding it with a horseradish peroxidase conjugate of MS-6E6, a anti-phospho-Syntide-2 monoclonal antibody, which then catalyses the conversion of the chromogenic substrate tetra-methylbenzidine (TMB) from a colourless solution to a blue solution (or yellow after the addition of stopping reagent). The colour is quantified by spectrophotometry and reflects the relative amount of CaM kinase II activity in the sample.
For kinetic analysis, the sample containing CaM kinase II is added to the wells in a similar fashion, and at varying times the reaction is stopped by the addition of a chelator, sodium thylenediaminetetraacetate (EDTA), and the amount of phosphorylated substrate determined as before.
The CycLex CaM kinase II Assay Kit is designed to determine non-isotopic kinetic analysis of CaM kinase II. Careful attention to extraction and purification methods and the assay protocol will provide the investigator with a reliable tool for the evaluation of CaM kinase II activity.
Product Information
CycLex® CaM-Kinase II Assay Kit (96 wells)
Related Products
CaM-kinase II Positive Control (3 units )
Anti-Phospho-Syntide-2 Monoclonal Antibody (100 μg)
Caltag Medsystems is the distributor of MBLI’s products in the UK and Ireland. If you have any questions about these products, please contact us.
To view MBL’s catalogue of Kinase Assay Kits, please click here.
References
Further Information
Ca2+/CaM-dependent protein kinase II (CaM kinase II) transduces elevated Ca2+ signals in cells to a number of target proteins, ranging from ion channels to transcriptional activators. CaM kinase II has autoregulatory properties that allow it to give a prolonged response to transient Ca2+ signals and to sense cellular Ca2+ oscillations. In neurons, CaM kinase II is highly expressed and localised with certain subcellular structures. Upon activation, it can translocate to excitatory synapses where it regulates a number of proteins involved in synaptic transmission and downstream signalling pathways.
Changes in intracellular calcium can display variable responses – ranging from highly localised transient elevations within subcellular structures (e.g. a dendritic spine of a neuron), to Ca2+ waves that spread throughout the cell, including the nucleus. The most ubiquitous calcium-sensing protein is Calmodulin (CaM), which contains four “EF” hand motifs with a high specificity for Ca2+. When bound, the Ca2+/CaM complex interacts and modulates the functionality of a large number of proteins including several Serine/Threonine protein kinases.
The CaM kinase II family is encoded by four genes (alpha, beta, gamma, and delta) that also exhibit alternative splicing. The gamma and delta isoforms are expressed in most tissues, whereas the alpha and beta isoforms are most prominent in neural tissues. The various CaM kinase II subunits are comprised of an N-terminal catalytic region, a central regulatory domain containing an autoinhibitory domain (AID) and Ca2+/CaM binding motif, a variable sequence, and the C-terminal subunit association domain.
In the absence of bound Ca2+/CaM, CaM kinase II is maintained in an inactive conformation because of an interaction of the AID with the catalytic domain of its own subunit. The Ca2+/CaM complex binds to a sequence that partially overlaps the AID, presumably causing a conformational change and thereby disrupting interaction of the AID with the catalytic domain, resulting in kinase activation. Interestingly, the sensitivity of CaM kinase II to activation by Ca2+/CaM depends on the subunit composition of the holoenzyme.
Measurement of CaM Kinase II Activity
The CycLex CaM kinase II Assay Kit uses a peroxidase coupled anti-sequence-specific phosphoserine monoclonal antibody as a reporter molecule in a 96-well ELISA format. This assay provides a non-isotopic, sensitive and specific method to detect CaM kinase II activity. The CycLex CaM kinase II Assay Kit is designed to accurately determine the presence and relative amount of CaM kinase II activity in purification column fractions, and for the non-isotopic kinetic analysis of CaM kinase II activity. Careful attention to extraction methods and the assay protocol will provide the investigator with a reliable tool for the evaluation of CaM kinase II.