Receptor tyrosine kinase assays

Details Receptor tyrosine kinase assays Receptor tyrosine kinase assays

C - Chemistry, Metallurgy

12

Q

C12Q 1/68 (2006.01) C12Q 1/48 (2006.01) C12N 15/85 (2006.01)

Patent

CA 2734895

Methods for detecting phosphorylation of receptor tyrosine kinases ("RTKs") upon activation are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of .beta.-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of .beta.-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a .beta.- galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.

L'invention concerne des procédés de détection de la phosphorylation de récepteurs tyrosine kinases ("RTK") lors de leur activation. Le procédé utilise des cellules comprenant deux produits de fusion: (1) une RTK fusionnée à un petit fragment de ß-galactosidase et (2) un peptide se liant à une phosphotyrosine fusionné au grand fragment de la ß-galactosidase, ces 2 fragments formant faiblement un complexe produisant une enzyme active, et éventuellement un gène hybride destiné à une kinase phosphorylant une RTK cytosolique, quand la RTK ne s'autophosphoryle pas. Pour détecter la phosphorylation, un substrat de ß-galactosidase est ajouté aux cellules, la formation d'un produit indiquant la présence d'une phosphorylation.

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