Kinases assays

Several excellent reviews have been written over the last decade highlighting the many different kinase assay formats available and their application to specific enzymes [20-22]. This section will only briefly review the current formats used within the HTS environment. The reader section consult the above-mentioned references for greater detail of each of the formats. 

Another popular assay format for kinase assays is the Lanthascreen. This format is a variation on the LANCE assay, but employs Tb as the cryptate. In this format N-terminally fluorescently tagged peptide substrate (acceptor) is phosphorylated by the kinase. Next, a phophospecific antibody which is labeled with terbium binds specifically to the phosphorylated product, placing the donor and acceptor in close proximity, generating a signal [25]. 

Ponzetto, C., Wadewitz, A. G., Pendergast, A. M., Witte, O. N., and Wolgemuth, D. J. (1989). P I50c ai,/is detected in the mouse male germ line by an in vitro kinase assay and is associated in haploid cells with a stage-specific phosphoprotein. Oncogene 4 685-690. 

Simard JR, Getlik M, Griitter C et al (2009) Development of a fluorescent-tagged kinase assay system for the detection and characterization of allosteric kinase inhibitors. J Am ChemSoc 131 13286-13296... 

Volume 200. Protein Phosphorylation (Part A Protein Kinases Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression)... 

Direct kinase assays can be performed for all of these enzymes following their immunoprecipitation from cell lysates (see protocols later). 

The activities of S6K1 or S6K2 may be assessed by direct (radioactive) kinase assay using a 32-amino acid peptide (Moule et al, 1995), 40 S ribosomal subunits (Chen and Blenis, 1990), or S6, expressed as a fusion protein with GST (Patti et al, 1998). 

Immunoprecipitation for protein kinase assays using peptide substrates... 

Immunoprecipitation for kinase assay against a protein substrate... 

S6 kinase assay using GST-S6 or 40 S subunits This is performed according to the general method described previously but using GST-S6 (Patti et ah, 1998) or 0.5 A260 units of 40 S ribosomal subunits per assay (Chen and Blenis, 1990). 

Fig. 12 Cartoon representing two versions of a kinase assay, (a) The tum-off version is a direct assay the extent of phosphorylation of the substrate is reflected in the binding of the quencher to the bead and hence to the quenching [88]. (b) The tum-on version is a competition assay increased phosphorylation of the substrate leads to it competing with the quencher in binding to the beads, and hence to increased emission [44]. Emissive polymers are represented by green lines quenched polymers by grey lines...

Rininsland F, Stankewicz C, Weatherford W, McBranch D (2005) High-throughput kinase assays with protein substrates using fluorescent polymer superquenching. BMC Biotechnol 5 16... 

Comparison of inhibition curves generated by four compounds following serial dilution in assay buffer containing 1% (v/v) DMSO (circles) and in neat DMSO (triangles) in a protein tyrosine kinase assay. 

Sills, M.A., Weiss, D., Pham, Q., Schweitzer, R., Wu, X., and Wu, J.J., Comparison of assay technologies for a tyrosine kinase assay generates different results in high throughput screening, /. Biomol. Screen., 7,191, 2002. 

Pommereau, A., Pap, E., and Kannt, A., Two simple and generic antibody-independent kinase assays comparison of a bioluminescent and a microfluidic assay format, /. Biomol. Screen., 9, 409, 2004. 

Turek-Etienne, T.C., Lei, M., Terracciano, J.S., Langsdorf, E.F., Bryant, R.W., Hart, R.F., and Horan, A.C., Use of red-shifted dyes in a fluorescence polarization AKT kinase assay for detection of biological activity in natural product extracts, /. Biomol. Screen., 9, 52,2004. 

Von, L.A., Kumpf, R., Menzel, S., Reulle, D., Griebel, R., Valler, M.J., and Buttner, F.H., Miniaturization and validation of a high-throughput serine kinase assay using the alpha screen platform, /. Biomol. Screen., 9, 719, 2004. 

In assays of enzyme activities a cofactor, but not a prosthetic group, can be easily lost from the enzyme by dilution during extraction or purification, or removed by agents that will bind the cofactor. For these reasons, an excess of cofactor is routinely added to the assay medium (e.g. in kinase assays) for the measurement of enzyme activity. 

Fig. 15.26 Visualization of the two sets of diversity elements arrayed around a single core. Triangles indicate inactive molecules in a kinase assay squares indicate synthesis failures circles indicate active kinase inhibitors within this set.

Figure 6.34 Elastomer-based microwell protein kinase assay array. (From Zhu, H. et al.. Nature Genetics, 26, 283-289, 2000. With permission.)...

An in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells OR an in vitro mouse lymphoma thymidine kinase assay... 

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