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Can I detect this probe using a UV/Vis spectrophotometer


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#1 cardosopedro

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Posted 19 February 2011 - 05:06 AM

Dear all,

I will try to measure the intracellular concentration of K+ using PBFI-AM. Does anyone here work with this probe? It is excited at 340/380nm and emits at 500 nm (I think).

I want to compare PBFI-loaded cells from two different cell types. Therefore, I do not need to determine the absolute concentration of K+. I only want to compare the two types of cells by fold change/arbitrary units.

I thought I would have to use a fluorometer to do the measurement, but then I was told that I could trace the spectrum of the PBFI-loaded cells using a UV/Vis spectrophotometer and then calculate the difference between the two cell types. Can someone advise me on this?


Please help.
Thanks.

#2 bob1

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Posted 19 February 2011 - 12:47 PM

Using a spec would only work if the PBFI-AM is changed into something else when bound to K+ - so that you can see the change in absorbance with molecular change. I personally don't think it is a viable technique, but it may be worth a try. I think it would work if you could excite it at 340/380 nm and then use the UV-vis spec to measure the emission curve.

#3 cardosopedro

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Posted 20 February 2011 - 04:03 AM

Thank you.

Here is something I found in a paper:
"Binding of K+ induces a 2.5-fold enhancement of fluorescence intensity. Furthermore, the excitation and emission maxima of PBFI shift to shorter wavelengths. The ratio of the fluorescence signals obtained by exciting PBFI at 340-345 nm and at 370-390 nm while monitoring the fluorescence at 450-550 nm is used to determine the intracellular K+ concentration."

I'm confused.
:unsure:

#4 bob1

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Posted 20 February 2011 - 04:31 PM

Basically this means that if the PBFI is bound to K+, it will excite more at 340 nm than it will at 370 nm, when compared to unbound PBFI, which should excite relatively more at the 370 nm wavelength.

You won't be able to detect this by spectroscopy unless you can excite and measure the spectrum at the same time... I think you need a fluorescent plate reader.




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