We are trying to find out whether increase or decrease of cytoplasmic calcium concentration affects the interaction of two proteins, using co-IP in HEK293T cells as a readout.
In different forums and papers I have found that to increase cytoplasmic Ca2+ levels in non-exciteable cells, there are three major categories of compounds. Ionophores (A23187, Ionomycin), Agonists of InsP3-PKC signalling (carbachol, ATP) and SERCA inhibitors(Thapsigargin, CPA).
However, different papers and different people on use different concentrations and durations of treatments. Since a lot of the drugs generate rather transient spikes, I realize that the timing is crucial here. Since, we do not have the resources for the optimisation of [Ca2+]-sensing dye imaging, I am also wondering whether there is a simpler way of roughly assessing [Ca2+], perhaps by by analyzing a marker protein/RNA using Western Blot or qPCR?
Thanks a lot!