Hi,
I was wondering if anyone would be able to recommend a simple way to test whether a any calcium binding has occurred with calmodulin (a purified protein sample purchased from calbiochem) in a solution?
Thank you very much
To test the purchased protein for bound calcium, you could denature and precipitate a sample of it, then test the supernatant for calcium using a calcium-sensing fluorescent dye.
The denaturation/precipitation method using perchloric acid followed by neutralization with potassium carbonate can be found in this paper:
http://www.jbc.org/content/265/8/4340.long
Care would be needed to avoid adding additional calcium by using the highest purity reagents.
A selection of calcium-sensing fluorescent dyes can be found here:
https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/indicators-for-ca2-mg2-zn2-and-other-metal-ions/fluorescent-ca2-indicators-excited-with-visible-light.html
Other methods for detecting calcium include ICP-MS and atomic absorption spectroscopy.
In the case of calmodulin changes in intrinsic fluorescence (excitation ~280 nm, emission 300-400 nm) are observed depending on calcium loading. The calcium free form (apo-CaM) has much lower fluorescence than the calcium bound calmodulin (holo-CaM). Complexed calcium can be removed by EGTA. Afterwards EGTA should be removed via several ultra filtration steps. One additional remark. Even if the purity of the purchased CaM is high (no other proteins in SDS-PAGE), the protein content is usually 60 - 75 %.
best
Björn
Fully Ca2+ -loaded CaM shows increased protease protection compared to Apo-CaM.
Please see figure S3 of the attached article.
Thank you very much for all your thorough help.
I realized that the protein was lyophilized in the presence of EDTA. Would that mean that the EDTA would have prevented calcium binding to calmodulin then? Or is it not a strong enough chelating agent to prevent trace calcium from binding?
At neutral or basic pH, the affinity of EDTA for Ca2+ is quite high, with dissociation constants in the pM range. So it is likely that free Ca2+ was bound up by EDTA if the conditions were appropriate.
Trace calcium? How much calcium did you have in your reconstitution buffer? If in your experiment you have to use only minimal required calcium to fully saturate the CaM with Ca++, then the company should be able to tell you the amount of EDTA and accordingly you can do maths to figure out the chelation factor. If the amount of Ca++ in your experiment is not the limiting factor then why you do not use excess of CaCl2 in your buffer. Or is it that you have already done the experiment and now you are wondering if your CaM was saturated with Ca++ in the concentration range you used?
The buffer pH was 7.4 so then EDTA should have a high affinity for calcium? I did not add any calcium chloride at all. I was hoping to examine a calcium-free calmodulin system and wanted to ensure that it was indeed calcium-free. Would it be acceptable to assume that trace calcium did not bind to the calmodulin since EDTA was present then?
Thank you for your help.
I agree that if EDTA was present at a meaningful concentration at neutral pH, trace calcium should have been unavailable for binding to calmodulin, which has a weaker affinity for calcium than EDTA does by 2 or 3 orders of magnitude.
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