I have transgenic lines of arabidopsis expressing aequorin in the tonoplast, and am using them to determine calcium fluxes in week-old seedlings after certain treatments. The issue I am running into is that I am not sure if I am normalizing my readings from the luminometer to the summation of all the luminescence readings taken during the experiment, or only after discharging the aequorin with 2M CaCl2 in 20% EtOH. And if so, do I integrate those readings with respect to time or simply leave it as the sum? Thank you for any help, I appreciate it!
You must incubate the transgenic lines of arabidopsis expressing apoaequorin in the tonoplast with coelenterazine (2.5 uM final conc. in dH2O) in dark conditions for the reconstitution of aequorin and avoid exposing them to any light before inserting them in the luminometer.
If the luminometer you're using is not able to dispense solutions and take measurements at different times, you'll have to measure the background noise first, then dispense the stimulus and take periodical measurements, finally discharge the maximum aequorin content and take the last measurements. Afterward you should use the background and the discharge for normalizing.
There are at least two isoforms of aequorin being used at the moment and each has its own calibration method.
I know that Prof. Knight from Durham University worked on one and the Prof.Votknecht from Bonn University worked on another.
I did my thesis with the latter and her group has a calculation sheet including macros for the calibration called CALIPSO, but I don't remember it being public.
I attach some useful literature:
Article Aequorin-based measurements of intracellular Ca-signatures i...
Chapter Aequorin-based measurements
Article Aequorin-Based Luminescence Imaging Reveals Stimulus- and Ti...
Article A toolset of aequorin expression vectors for in planta studi...
Article The ins and outs of Ca(2+) in plant endomembrane trafficking
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