I have a calcium-dependent protein. I should treat my cells with that but I don't know which buffer is the best. Someone advised me to use a ca-EGTA buffer, but I think it is harmful for my cells. Any help will be appreciated.
What is the goal of the experiment and which are the cells u want to treat?
U want to make the protein work or u want to have control over calcium levels? +
If u want to have controlled activation by calcium level modification, then may be u can use low-calcium DMEM (commercially available) and supplement with different amount of Calcium.
Dear Manoj Balakrishna Menon
the goal of the project is examination of the protein effect on the cells. my protein has calcium in its structure and the efficiency of that is dependent to calcium. i need a buffer which could save the structure and function of the protein but has no toxicity for the cells, pleas help me, i really don't know what should i do.
i forgot to say. my cell line is HS578T. bclc (basal like breast cancer)
The normal Calcium levels in DMEM supplemented with 10% FCS (recommented for ur cells) is somewhere between 1.5- 2mM. This amount of calcium in adherent cell culture medium helps the adherent junction proteins and other matrix proteins to function properly and should be enough for any protein which u add (I assume isolated from a recombinant source).
If you isolated this protein from a biological source, I would assume that it is already co-ordinated with calcium and u just need to maintain calcium levels to prevent dissociation. Since the intracellular or serum calcium levels are usually below this mM concentration, I dont think u will have any problem. As I mentioned before, u can use serum free calcium free medium as control, if ur assay demands it (for short term treatment!!). Hope this helps. Good Luck.
Manoj Balakrishna Menon
thank you very much. i'll try your advises.
best
Dear Najme
our research group has been used an adenovirus Ad-Ip3-buffer-NLS or Ad-Ip3-buffer-NES to buffer Calcium from nucleus or cytosol, respectively. Other possibility is transfect your cell with PV-NLS or PV-NES plasmid that is easy to buy them from Addgene. Those strategy are convenient because you can found out if the effect happens (inhibiting or stimulating) by subcellular calcium. Please, find in attachment some papers about.
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