Characterization of a biological detector cell for quantitation of inositol 1,4,5-trisphosphate.
Prior strategies to measure inositol 1,4,5-trisphosphate (IP(3)) 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 IP(3) 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 IP(3)-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 flow in the capillary, the concentration of free Ca(2+) 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 IP(3) 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.
Luzzi
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V
Department of Physiology and Biophysics, University of California at Irvine, Irvine, California 92697-4560, USA.
Murtazina
D
D
Allbritton
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NL
D000818:Animals; D015374:Biosensing Techniques; D002460:Cell Line; D019075:Electrophoresis, Capillary / Q000379:methods*; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis*
D015544:Inositol 1,4,5-Trisphosphate