I have some compounds expected to be inhibitors of tyrosine kinases, and i want to test their inhibitory action in vitro by ELISA. i used the cell lysis buffer recommended by the kits and stimulated them using PMA 100ng/ml but i get no results for the assay.
how can i stimulate tyrosine kinases (VEGFR2, PDGF b, cKIT and FLT-3) in HeLa, MCF-7 and HCT116?
hi
This article may help you
http://www.ncbi.nlm.nih.gov/pubmed/12376368
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358446/
good luck.
Mr Hadavi, thank you for your help. i read the two articles but non of them applies to my research. i am testing these compounds on cancer cells but not normal cells. i want to know can i induce the expression of tyrosine kinases in the mentioned cell lines. they do already express them but when i test the cells (untreated and unstimulated) i got no results. this is the problem i have now. how can i find a stimulator for the VEGFR2?
You could try with Ionomycin to stimulate VEGFR2. Some cell lines at distinct experimental conditions differ with their functions after certain stimulants. You can also try at different time points, early time point induce the trafficking receptors which is already synthesized, de novo synthesized receptors take longer time, try with kinetics. One more suggestion, if non of the stimulants work in your condition, change the condition media, by reducing the FBS concentration and also try with VEGF ligand.
In general, the VEGFR2 is the major VEGF receptor in cancers so no extra stimulus is required for its expression. however one can try cloning the gene and can be transfected into the cell line.. however it will be hectic.
for your refernce
Complementary DNA clones for rhVEGF-C, isolated from a human osteoclast cDNA library, were assembled into the pcDNAI/Amp mammalian expression vector that includes a cytomegalovirus promoter/enhancer to drive gene expression. This plasmid construct was named pcVEGF-C. The plasmid pGSVLacZ (courtesy of Dr. C. Bonnerot), containing a nuclear targeted β-galactosidase sequence coupled to the SV40 early promoter, was used for control transfection experiments.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852989/
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