Hi Min,

A few things of the top of my head, the list is not exhaustive...

Firstly, what is the pH of the medium? If it is above pH 7 then calcium could precipitate with some of the small molecules in the medium. So the calcium concentration is not actually increased and you may have removed some other important metabolites from the medium

Secondly, the calcium is added outside the cell. It does not follow that the calcium concentrations will linearly increase inside the cells. Or inside the nucleus of the cells (the calcium has to cross two membranes to get there). Indeed, the cells will be working hard to maintain a ~100 nM free calcium concentration inside the cells so all you are doing is stressing them but not actually increasing the free calcium concentrations in the cytoplasm or nucleus. Do you see the same level of cell growth in high Ca media? Are the cells healthy?

Thirdly, do you know if your CaMBP binds to Ca-CaM or apo-CaM? As CaM does bind to some proteins in the metal-free form and is released upon binding to calcium (in which case your QPCR results would be perfect! - except some of the other 'solutions' stated here would be brought up as reviewers comments if you tried to publish)

Also, there are many different EF-hand proteins expressed inside cells. After much research, some calmodulin-binding proteins are often found to be more likely bound by different members of the family. Also, plants are special as they have several variants of CaM. Indeed, I think we have to be careful applying knowledge on human/mammalian EF-hand proteins into plant systems.

Fourthly, CaM is an important protein but so are many others. It is possible that regulation is through other factors than calcium. Calcium spikes that govern CaM interactions with other proteins last milliseconds - this is too fast to directly affect protein expression.

Fifthly It should be remembered that calcium spikes are normal physiological events. CaM binds to many proteins in a calcium-dependent way. While biochemists measure binding affinities, few study kinetics. Beat Schwaller has a few good papers on this topic. Basically, you can have two calcium-binding proteins with the same binding affinity but if one has 10-fold faster kinetics then that protein will become calcium-bound before the other. Remember that there are many other CaM-binding proteins in your cells as well as many calcium-binding, EF-hand proteins other than CaM. The calcium stress that you invoke is more likely to induce the up-regulation of calcium-buffer proteins to maintain the ~100 nM free Ca concentration rather than CaM, which is a calcium sensor.

These kind of experiments are very tough. If you get a positive result, then great. However, there are so many complicated factors working in cell biology to result in negative results. Don't be discouraged, try to think of another approach to study the system. Personally, I am a bit old-school and would try to express and purify the protein to do some classic biochemical studies before trying more complex cell biology experiments.

regards,

patrick groves

Dear Dr. Patrick Groves,

 Thank you so much for your kind answers and valuable comments.

 Normally the pH of MS medium is 5.8. And we always prepare MS with 3 mM of CaCl2. My CaMBP gene named NPG1 has been approved in binding to CaM in in a calcium-dependent way (Golovkin and Reddy, 2003). NPG1 has been verified as a pollen-specific CaMBP. However, the expression level of NPG1 was also detected during somatic embryogenesis of longan (Lai & Lin, 2013). For me I just want to test how NPG1 responses to the different concentrations of Ca. But after getting the results, I need to find the reason.

 I do appreciate your kindness in trying to help me.

 With best regards,

Min