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METHODS IN CELL PHYSIOLOGY

Xenopus oocyte plasma membrane sheets for FRET analysis

Departments of ¹Physiology and ²Medicine and Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California, Los Angeles, California

Submitted 14 August 2006 ; accepted in final form 7 December 2006

Plasma membrane sheets from Xenopus oocytes have been isolated for use in fluorescence resonance energy transfer (FRET) measurements. This system has the following advantages: 1) fluorescent recordings from a large surface area to maximize the signal-to-noise ratio, 2) reduction in background fluorescence from proteins retained in intracellular compartments, and 3) access to the cytoplasmic surface of the plasma membrane for rapid solution changes. To demonstrate the utility of this approach, we have examined a previously published FRET-based Ca²⁺ sensor, namely, the Cameleon-PM. This construct targets to the plasma membrane and, upon various Ca²⁺ additions to the cytoplasmic face of the membrane, shows ratiometric FRET changes. From the ratiometric changes recorded, an apparent Ca²⁺ affinity of 1.65 µM was determined. Thus preparation of Xenopus oocyte plasma membrane sheets and FRET measurements demonstrates all three of the advantages outlined above.

fluorescence resonance energy transfer

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