How to test if a compound is entering cells? - (Aug/25/2011 )

I'm characterizing a new compound from Maybridge that is supposed to inhibit my kinase, but so far it isn't doing anything at up to 1000X the reported Ki in cell-based assays. I plan to do some in vitro kinase assays to see if the thing works, but I also would like to determine if this compound is entering into cells. The Maybridge datasheet indicates "OK" for human intestinal absorption and "+/-" for BBB, so presumably they have done some testing. Does anyone have a clever assay to test if a chemical is entering into cells? Are there ways to predict if a chemical will enter cells? I suppose I could get lucky and the compound might be fluorescent, which would make tracking it easy. Does anyone know a way to predict which chemicals will exhibit fluorescence?

I am not sure of your definition of entering the cells, whether if the compound is located in cell membrane is satisfactory for you. But you could harvest your cells and extract your compound, and run it through the HPLC. To determine if your compound is fluorescence, you can try spectrofluorometer and run a scan? Or HPLC coupled with fluorescence detector may also run spectrum scan and tell you the optimum emission and excitation wavelength. If you define 'entering cells' by being present in cytosol, then may be gradient centrifugation where you can lyse the cells and obtain the cytosol only will work?

Just a thought, do you know if your compound is detectable by absorbance/fluorescence from the cell media i.e. if you take your cell media as a blank and cell media + compound at some wavelength would there be a change? If yes maybe you could follow that change simply by checking media before cell growth and after (maybe use a scan rather than a fixed wavelength readout). If the compound has a specific signature I can imagine this should work.

Hola one way to detect kinase inhibitors is to see the kinase forms in Western blot. for instance here to see if any drug has any effect on some kinases we see kinase and phospho-kinase (inactive) before and after treatment.Moreover we see some kinases downstream in the pathway of the kinase in study. (bad/p-bad, akt/p-akt, etc). Buena suerte