Can I detect this probe using a UV/Vis spectrophotometer
Posted 19 February 2011 - 05:06 AM
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?
Posted 19 February 2011 - 12:47 PM
Posted 20 February 2011 - 04:03 AM
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."
Posted 20 February 2011 - 04:31 PM
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.