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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!

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As far as I know, microscopy (be it with dyes like fluor or with genome-encoded calcium indicators) is really your only good bet, especially for getting quantitative sensitivity, which I think is what you're looking for. Changes in cytosolic calcium are incredibly dynamic both in time and space, which is something to consider when asking how calcium affects your two proteins.

I do know of an example where investigators transferred cells to buffers with different Ca2+ concentrations and permeablized them with a mild detergent to raise calcium levels (Borges-Pereira et al 2015). I'm not sure if that would work for your system, but it's worth taking a look, as I think it raises the levels pretty gently and steadily, and you have a better idea of what your intracellular concentration is going to be without directly visualizing it. Considering you won't be able to tell the Ca2+ concentration at any given time without microscopy, things like SOCE and intracellular chelation rates might be important, so you'll want to watch out for when after treatment you start your assay, perhaps trying a few different time points

Hope that was of some help!

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