...how to know the free Ca++ in the pipette?

8 posts / 0 new
Last post
Wanderermann
Wanderermann's picture
...how to know the free Ca++ in the pipette?

 Hi

How can I know how much is the free calcium in the recording pipette when both, calcium and EGTA are used? For example when the pipette solution have 1mM CaCl2 and 10mM EGTA, or when it have 0.5mM CaCl2 and 5mM EGTA.

Thanks.

Regards. 

Simon8
Simon8's picture
There are calculators that

There are calculators that can give you exact numbers for this, for example:
http://www.stanford.edu/~cpatton/CaEGTA-TS.htm

Wanderermann
Wanderermann's picture
 ...using that program, if

 ...using that program, if the amount of EGTA is 5mM and calcium is 0.5mM, the free calcium in the solution will be 10,31nM (0,01µM), am I right? I saw that in most of the cases this is the EGTA-calcium combination that people use in the recording pipette; so, the free intracellular calcium should be normally so low?

Thanks.

Simon8
Simon8's picture
Yes, it should be in the

Yes, it should be in the nanomolar Range. My intra has only 5nM free Ca2+, according to the calculator I posted. However I am not sure, what " Ionic = N" means.

Wanderermann
Wanderermann's picture
 ...me neither, I don´t know

 ...me neither, I don´t know what "Ionic = N" means. Here there is one paper where they said the pipette contain 0.5mM EGTA and then they add 1mM CaCl2 to obtain 275nM free calcium. Some idea why? How is calculated? Or is maybe not right?  

The paper:
http://www.jbc.org/content/286/3/2365

TMEM16A Inhibitors Reveal TMEM16A as a Minor Component of Calcium-activated Chloride Channel Conductance in Airway and Intestinal Epithelial Cells
Wan NamkungPuay-Wah Phuan and A. S. Verkman
January 21, 2011 The Journal of Biological Chemistry, 286, 2365-2374.

Simon8
Simon8's picture
Hmm from the Calculator I get

Hmm from the Calculator I get around 500nM (0.00050076M).

However, I have no Idea how it is calculated. There my be different ways? Or one of the sources (the paper or the calculator) is wrong.

Wanderermann
Wanderermann's picture
 ...I think you mean 500µM

 ...I think you mean 500µM instead 500nM (taking pH 7.3 and room temperature 23°C)

Simon8
Simon8's picture
Hmm of course. Missed the

Hmm of course. Missed the mikros in between
0.5mM EGTA = 0.0005M
1mM total Ca = 0.001M

The calculator then gives 0.00050016M Free Ca for 23°C and pH7.3. That is 0.5mM = 500muM.

That is of course very much. And not at all in the range of the given value...