Hi guys. Does anyone have experience with the radiometric dye SNARF? I am trying to do a calibration step but I think that I am doing something wrong because in the end I cannot get meaningful pH values (I am getting negative values).
When calculating the concentration of hydrogen ions, there is a term:
(R-RB)/(RA-R)
where R= the ratio measured at a given pH, RB is the ratio at the basic limiting pH, and RA is the ratio at the acidic limiting pH. R needs to be larger then the RB term and smaller than RA or you'll get negative numbers.
This dye seems to have a pH range of 6 to 9 (by the emission spectrum) and trying to use the dye outside of that range would be suspect. Fisher Scientific mentions a range of pH 7-8. In theory, one could use this dye as an excitation ratio, but the excitation at wavelengths smaller than the isobestic wavelength doesn't change much as a function of pH, so it is much better to use this dye as an emission dye.
Make sure you aren't seeing stray excitation light, make sure you have the correct dichroic filter (if doing microscopy), and make sure you don't have stray light from other sources. Also consider a look-up table as well. Has the cell had enough time to cleave the AM ester?
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