LOCALIZED SAMPLING OF CYTOPLASM FROM XENOPUS OOCYTES FOR CAPILLARY ELECTROPHORESIS

Continued progress in cellular physiology requires new measurement str ategies which can be applied to solitary cells, Since many cellular si gnaling pathways act on time scales of a few seconds, there is a criti cal need for single-cell techniques with subsecond time resolution. Ca pillary electrophoresis shows great promise as a tool for the analysis of individual cells, In the present work, we describe a technique to load a capillary with picoliter to nanoliter volumes of cytoplasm and initiate electrophoresis in less than 500 ms, When cytoplasm was sampl ed from a Xenopus laevis oocyte previously loaded with fluorescein, ca lcium green, or a mixture of the two fluorophores, their fluorescent p eaks were readily identifiable on the electropherogram, Since the volu me of cytoplasm (less than or equal to 30 nL) loaded into the capillar y was much smaller than the 1 mu L oocyte volume, spatially localized biochemical measurements were also possible. To demonstrate the utilit y of this new technique, the activity of the enzyme beta-galactosidase was measured in small regions of the Xenopus oocyte. Subcellular, sub second sampling of oocyte cytoplasm will enable biochemical measuremen ts with the resolution required to understand many cellular signal tra nsduction pathways.