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| {{Mainpage orobopedia}}
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| {{Product
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| |description='''O2k-TPP<sup>+</sup> and Ca<sup>2+</sup> ISE-Module''': 2 potentiometric ion-selective electrodes ([[ISE]]), ISE-Service Kit and 2 [[Stopper\white PVDF\angular Shaft\side+6.2+2.6 mm Port|PVDF-Stoppers]], supported by the [[O2k-Core]].
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| |product id=12300-01
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| |product type=O2k, O2k-Module, MultiSensor, Stopper, Catalogue
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| |info=[http://www.oroboros.at/?O2k-MultiSensor O2k-Multisensor @OROBOROS], [[MiPNet15.03|ISE-Manual]], [[Tetraphenylphosphonium]]
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| |product image=[[File:O2k-TPP-ISE.jpg|180px|right|link=]]
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| }}
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| == '''O2k-TPP+ and Ca2+ ISE-Module ''consists of'':''' == | | {{Technical support}} |
| {{#ask: mainlabel=Title|[[Category:Products]] | [[Product type::O2k-TPP-Ca ISE-Module]]
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| | |description=[[File:TPP new.jpg|180px|right]]'''O2k-TPP<sup>+</sup> ISE-Module''': Potentiometric ion-selective electrodes for measurement of mitochondrial membrane potential |
| |sort=product id
| | |info= [https://www.oroboros.at/index.php/product/o2k-tpp-ise-module/ '''Product details and purchase information'''] |
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| __TOC__ | | __TOC__ |
| = General = | | == Additional resources == |
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| The OROBOROS [http://www.oroboros.at/?O2k-MultiSensor ISE system] is designed with replaceable membranes making it possible to measure different ions such as TPP<sup>+</sup> or Ca<sup>2+</sup> with the same electrode housing.
| | :::: »''O2k-Manual:'' [[MiPNet15.03 O2k-MultiSensor-ISE]] |
| | :::: »''O2k-Procedures:'' [[Tetraphenylphosphonium#O2k-technical_support |TPP: O2k-technical_support]][[Image:O2k-TPP-ISE.jpg|180px|right]] |
| | :::: »''O2k-Specifications:'' [[MiPNet15.08 TPP electrode]] |
| | :::: »[[O2k-Catalogue: O2k-Modules| O2k-Modules]] |
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| == O2k-Core and O2k Series A-E ==
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| For the Oxygraph-2k Series A-E, the '''O2k-TPP<sup>+</sup> and Ca<sup>2+</sup> ISE-Module''' is supported by the [[O2k-Core]] and by all O2k instruments with the [[O2k-MultiSensor|O2k-MultiSensor electronic upgrade]].
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| = TPP /TPMP electrode =
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| The main purpose of the O2k-TPP+ and Ca2+ ISE-Module is to measure [[Mitochondrial membrane potential]] via the TPP or TPMP method.
| | == Calcium electrode == |
| | »''Membranes:'' [[ISE-Ca2+ Membranes]] |
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| The first publication using the O2k-TPP+ and Ca2+ ISE-Module for this purpose was [[Brown 2011 Am J Physiol Regul Integr Comp Physiol]]
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| | {{Keywords: Force and membrane potential}} |
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| = Calcium electrode =
| | {{MitoPedia O2k and high-resolution respirometry |
| | | |mitopedia O2k and high-resolution respirometry=O2k hardware, O2k-Open Support |
| A Ca<sup>2+</sup> selective membrane with a composition similar to the one described by Simon and coworkers <ref>Schefer U, Ammann D, Pretsch E, Oesch U, Simon W (1986) Neutral carrier based Ca<sup>2+</sup>-selective electrode with detection limit in the sub-nanomolar range. Analyt Chem 58: 2282-2285.</ref> has been tested with the OROBOROS ISE system and detection limits of less than 10 nM were achieved in Ca<sup>2+</sup> buffered calibration solutions. There may be further optimizations of the membrane composition to ideally fit the the typical applications of OROBOROS customers. However, the main obstacle in establishing useful Ca<sup>2+</sup> measurements appears to be not the Ca<sup>2+</sup> sensitive membrane but the methodology of its application in a medium simulating intracellular conditions.
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| | {{MitoPedia topics}} |
| ''Note: The operation of a Ca<sup>2+</sup> electrode at typically high extracellular Ca<sup>2+</sup> concentrations is straightforward and devoid of the difficulties described here.'' These difficulties include both the calibration of the Ca<sup>2+</sup> electrode and the planning of a biological experiment to use it.
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| == Operation of the Ca<sup>2+</sup> electrode ==
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| === General ===
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| The general operation (assembly, ...) of the Oroboros ISE system is described in [http://www.oroboros.at/?O2k-MultiSensor MiPNet15.03].
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| While working with the pX channel please always observe the guidelines for avoiding damage to the electronics by [[ESD]].
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| === Calculation of free Ca<sup>2+</sup> concentrations ===
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| To work at physiological Ca<sup>2+</sup> concentrations requires usually Ca<sup>2+</sup> buffering by chelating agents. The calculation of ''c''(Ca<sup>2+</sup>)<sub>free</sub> in such solutions is quite complicated and the results depend i.a on ionic strength, temperature, and - very strongly - on the pH. Tools for such calculations are available on-line at Chris Pattons MAXCHELATOR Page [http://maxchelator.stanford.edu/ maxchelator.stanford.edu]. Many calculations, published or implemented in programs, depend on a single set of data compiled by Martell and Smith <ref>Martell AE, Smith RM (1989) Critical Stability Constants, 1: Amino Acids. Plenum Press</ref>.
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| Note that inspite of the availability of easy to use software tools the calculation of free Ca<sup>2+</sup> concentration is not simple. The accuracy of these calculations is severely limited by several factors:
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| * Many underlying thermodynamic constants may not be known precisely
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| * Some thermodynamic constants necessary for adequate temperature correction may not be known at all
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| * It may be difficult to set the experimental parameters pH and ionic strength with the necessary precision
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| === Inner filling solution === | |
| The following inner filling solution is used:
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| CaCl<sub>2</sub>: 10 mM
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| EDTA: 50 mM
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| pH adjusted to 8.5 with KOH
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| === Conditioning ===
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| Conditioning of the membrane is a controversial topic. If any conditioning is done, the used free Ca<sup>2+</sup> concentration should probable not be much higher than the highest expected concentration during applications of the electrode.
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| === Calibration ===
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| As a potentiometric method, the Ca<sup>2+</sup> electrode delivers a signal that is (in the working range) linear to the logarithm of the free Ca<sup>2+</sup> concentration. Therefore, the electrode is calibrated by plotting electrode signal vs. logarithm of the free Ca<sup>2+</sup> concentration. Calibration of the Ca<sup>2+</sup> electrode at low (< 1µM) Ca<sup>2+</sup> levels is typically done by exposing the electrode to a series of Ca<sup>2+</sup> calibration buffers. Each calibration solution typically contains a Ca<sup>2+</sup> chelating agent, a pH buffer, CaCl<sub>2</sub>, and a salt (KCl) to adjust ionic strength. For suggested compositions see the literature in the reading list below. Examples from the literature may then be adapted to individual needs using the software tools mentioned above. The pH of all solutions have to be adjusted very carefully. For calibrations spanning a large range (several orders of magnitude) of free Ca<sup>2+</sup> concentrations it is necessary to use calibration buffers with very different compositions. The nonlinearity of the electrode response in such calibrations may be mainly due to the difficulties in calculating the free Ca<sup>2+</sup> concentration.
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| == Application in biological experiments ==
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| You could help us in the development by explaining what you would like to do/ see in a Ca<sup>2+</sup> experiment and what you expect from the electrode: what ca concentrations do you want to measure, Ca<sup>2+</sup> release or uptake? what total concentration change in a 2 ml chamber (!) do you expect? Do you want to measure in a Ca buffered medium? (decreased sensitivity to changes) or without Ca<sup>2+</sup> buffering but then how do you get to physiological Ca<sup>2+</sup> concentration? Do you want to measure at physiological Ca<sup>2+</sup> concentrations?.......
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| * Please add your comments in the [[Talk:O2k-TPP+ and Ca2+ ISE-Module|Discussion page]]. Pease contact instruments@oroboros.at to recive an account.
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| * user feedback: FCCP may be incompatibe with the function of the [[ISE-Ca2+ Membranes]]. [[User:Fasching Mario|Fasching Mario]] 11:45, 2 November 2012 (CET)
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| == Reading list ==
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| We have complied a short reading list that may be of interest to those planing to to do Ca<sup>2+</sup> measurements, with special emphasis on references describing the preparation of Ca<sup>2+</sup> calibration buffers.
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| [[Media:Ca_reading_list.pdf|Ca measurement reading list]]
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| == References ==
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| <references/>
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| {{#set:Technical service=Ca}}
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| <br> {{Technical service}}
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