Xenopus oocyte plasma membrane sheets for FRET analysis.
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 Ca(2+) sensor, namely, the Cameleon-PM. This construct targets to the plasma membrane and, upon various Ca(2+) additions to the cytoplasmic face of the membrane, shows ratiometric FRET changes. From the ratiometric changes recorded, an apparent Ca(2+) affinity of 1.65 microM was determined. Thus preparation of Xenopus oocyte plasma membrane sheets and FRET measurements demonstrates all three of the advantages outlined above.
Ottolia
Michela
M
Cardiovascular Research Laboratories, MRL 3-645, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1760. sjohn@mednet.ucla.edu).
Philipson
Kenneth D
KD
John
Scott
S
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006056:Golgi Apparatus / Q000378:metabolism; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D002118:Calcium