The reversible addition of phosphate groups to proteins is important for the transduction of signals within eukaryotic cells, and as a result, protein phosphorylation and dephosphorylation regulate many diverse cellular processes. Protein kinases that phosphorylate proteins in eukaryotic cells belong to a large family of enzymes, all of which contain a similar 250 amino acid catalytic (kinase) domain. Major families include cyclin-dependent kinases, MAP kinases, tyrosine kinases, and receptor serine kinases. As the number of known protein kinases has increased at an ever-increasing rate, it has become more challenging to determine which protein kinases interact with which substrates in the cell. Recently, because of their importance in many cellular events, protein kinases have become likely targets within the drug discovery community. To aid the discovery of new protein kinases, and elucidate specific kinase activity in the presence of candidate drug compounds, Whatman have produced a Protein Kinase Assay filterplate that enables for multi-sample evaluation. Protien kinase enzymes transfer the terminal phosphate group of ATP to a specific amino acid sequence of a target protein (substrate). If the kinase reactions are carried out in the presence of radio-labeled ATP, the resultant phosphorylated substrate will in turn become labeled. Measuring the labeled substrate by liquid scintillation counting (LSC) will give an indication of kinase activity. The key to a successful separations based procedure such as a kinase assay, where un-phosphorylated substrate is removed from phosphorylated (radio-labeled) substrate, is the ability to capture the component of choice very specifically. The Protein Kinase Assay filterplate incorporates a P81 phoshocellulosic filter at the bottom of each of its 96 wells. P81 specifically binds phosphorylated substrate, allowing unlabeled substrate and unincorporated ATP to flow through - this results in low non-specific background noise and thus high sensitivity. The Protein Kinase Filterplate is manufactured to international SBS standards in rigid white polystyrene designed to eliminate absorption problems during LSC.

• Discrete P81 filter at the bottom of each well - specific to phosphorylated labeled substrate.
• 96 wells - multiple sample applications.
• Evaluation of many protein kinase families - does not require specific antibodies.
• Short drip directors that can facilitate non-cross talk collection of radioactive
• waste filtrate.

• Rapid evaluation of multiple samples.
• Automation and liquid handling compatible.
• Suitable for use with all 96 well LSC plate readers.

 
    There are many families of protein kinases, and therefore the assessment of their activity requires slightly differing protocols and procedures. All kinase activity assessment however, can be carried out using the filter plate, no matter what the family.

For demonstration a protein kinase C (PKC) procedure is shown:

    Assay PKC activity for 2 minutes at 30C in a 70ul reaction containing 20mM HEPES, pH 7.4, 1.0mM DTT, 1mg/ml histone, 10mM MgCl2 and 0.15mM [ðg-32P]-ATP (0.05Ci/ml) in the presence and absence of 200ug/ml phosphatidyl serine, 10ug/ml DAG and 0.1mM CaCl2. Terminate the reaction with 1.5% phosphoric acid and then apply directly to the protein kinase assay filter plate. Incubate within the wells for 5 minutes and then wash 4 times, five minutes each, with 0.5% phosphoric acid - using a vacuum to waste manifold. When washed, continue vacuum for 10 minutes to dry the filter, and then count by LSC with addition of 100-200ul of scintillant per well.
 

ORDERING INFORMATION

Catalog No.	Description	Units/Case
921412	350µl, 96 well Whatman P81 filterplate	50
981801	Vacuum to waste manifold	1