According to the companies Description, BAPTA is Ca2+ chelator exhibiting a 105-fold greater affinity for Ca2+ (Kd = 110 nM) than for Mg2+. After BAPTA complexes with Ca2+, the UV spectrum shifts from 254 to 279 nm at pH >6.0. Useful for spectrophotometric monitoring of extracellular Ca2+ levels, especially transient phenomena, because of its insensitivity to pH.
I have used 50mM Tris Hcl (pH:9) for preparing 10mM BAPTA solution. then I used this buffer for determining a standard curve at different ca2+ concentration (10^-8 M to 10^-4 M). but I only saw a sharp absorbance at ~230nm which didn't change significantly following Ca2+ addition. Is there any body who worked previously with this compound? any advice will be greatly appreciated
(please note that I am trying to concentrate free Ca2+ ion by an indicator to determine ligand+protein binding)
I have no personal experience with this way of measuring Ca. But notice that it would hardly work in extracellular environment. Typical extracellular Ca is ~1 mM, and BAPTA affinity is 0.1 microM. This means that 99.99% BAPTA is already Ca-bound, and one hardly can expect noticeable spectral changes with changing Ca. Also, possible impurities of the salts used for making the perfusion medium well could bring Ca concentration up to ~10 microM. Thus in nominally-Ca-free medium , 99% BAPTA could be in Ca-bound form.
Dear Vector
Thanks for your reply. But I have made Ca2+ ion standard solutions in the range 10 nM to 10 mM !
The standard solutions would not help anyway since I guess your normal working extracellular medium Ca would be in saturating range for BAPTA. The lack of spectral effects between 10 nM is 10 mM is and enigma indeed. Then the question is, what was the source of your Ca standards. If you made them yourself, in well could be contaminated up to micromolar range.
Hello, I'm curious if you ever figured out the problem (I see that your question was posted over a year ago). Could it be that more Ca2+ was present in your buffer than you realized? I find that the "deionized" milliQ type of water still has uM Ca2+ and Mg2+ content which will drastically throw off the concentrations, especially if you wish to monitor in that range or below. In that case, if you can, try to start with a calcium-free water, and you can have your water analyzed by ICP-OES to find out its metal content.
Chrystal Archer, what made you come to that conclusion about the Ca2+ in the milliq water? I am struggling to get my protein calcium free, I think I have excluded most reasons but the milliq contamination.
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