Characterization of a biological detector cell for quantitation of inositol 1,4,5-trisphosphate

Prior strategies to measure inositol 1,4,5-trisphosphate (IP3) in single cells either have been qualitative or have had a limited spatial resolution. Capillary electrophoresis combined with a biological detector cell has been used to quantitate IF, in small regions of a Xenopus oocyte, To improve the detection limits of this method, we elucidated the experimental parameters which influenced the sensitivity and reliability of the IP3-detector cell coupled to capillary electrophoresis, The variables which influenced the detector cell were the magnitude of the voltage drop across the detector cell, the duration of this voltage drop, the direction of fluid how in the capillary, the concentration of free Ca2+ around the detector cell, and the presence of protease inhibitors during permeabilization of the detector cell. For the sample volumes imposed by the capillary diameter, the detector cell acted primarily as an IF, mass detector rather than a concentration detector. Characterization of the experimental variables influencing the sensitivity and reliability of this detector cell has the potential to enhance other analyte measurements performed by mating capillary electrophoresis with a biological detector cell. (C) 2000 Academic Press.