Haspin Cellular Phosphorylation Assay (intracellular kinase activity assay) for compound screening and profiling in intact cells
Haspin (GSG2) represents a serine/threonine-protein kinase that specifically phosphorylates histone H3 at Thr3 during mitosis. Haspin associates with the chromosomes and thereby positions and activates Aurora B and other components of the chromosomal passenger complex (CPC). This activity ensures proper chromosome alignment and segregation and normal progression through the cell cycle. Haspin inhibitors such as CHR6494 have been shown to cause mitotic catastrophe in cancer cell lines such as HT-29 and to induce tumor regression in vivo, suggesting Haspin as promising target in tumor therapy.
Using HT-29 colon carcinoma cells, this cellular screening system monitors the phosphorylation level of HistoneH3 at Thr3, a direct downstream target of endogenous Haspin / GSG2. To boost the phospho-Histone-H3 signal, phosphatase inhibitor Calyculin is added prior to cell lysis. Using this assay, we could successfully show the inhibitory effect of CHR6494 on the levels of phospho-Histone-H3-Thr3 which was quantitated via direct ELISA (see. Fig.1).
Substrate phosphorylation as a readout of intracellular kinase activity via ELISA
More information can be found on our website Cellular Phosphorylation Assay Services.
Reference compound IC50 for Haspin
The cognate Haspin inhibitor CHR6494 blocks Haspin and potently inhibits the cellular phospho-Histone-H3-Thr³ signal. The graph shows the result of a representative experiment.