NR AXRX
AU Madeira,C.; Ferreira,J.A.B.; Andrade,S.; Costa,S.M.B.; Cabrita,G.J.M.; Melo,E.P.
TI Probing the Prion Protein at the Cell Surface with FLIM and FRET Measurements
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.42
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB The prion protein (PrP) is being expressed in N2a-BOS cells fused to CFP (cyan fluorescent variant of GFP) and YFP (yellow variant of GFP). PrP in mammals is GPIanchored (glycosylphosphatidylinositol) to the membrane and to have the protein attached to the membrane the signal to GPI-anchor was cloned downstream to the FP to attach the fusion protein to the cell surface. A clone selection protocol was optimized in order to obtain stable N2a clones expressing the fusion proteins. Cells were cultured on glass cover-slips to allow fluorescence images to be recorded. The fusion proteins at the cell surface were detected through the intrinsic fluorescence of the FP and the integrity of the fusion will be verified by western-blotting. Fluorescence lifetime imaging microscopy (FLIM) will be used to probe PrP at the cell surface. FLIM will be combined with fluorescence resonance energy transfer (FRET) measurements for the pair CFP-YFP. The combination of FLIM and FRET provides high spatial (nanometer) and temporal (nanosecond) resolution and is expected to give insight into conformational changes of PrP and to detect PrP-PrP interactions at the cell surface.
AD C. Madeira, G.J.M. Cabrita, Instituto Superior Técnico, Institute for Biotechnology and Bioengineering, Portugal; J.A.B. Ferreira, S. Andrade, S.M.B. Costa, Instituto Superior Técnico, Centro de Química Estrutural, Complexo I, Portugal; E.P. Melo, Centro de Biomedicina Molecular e Estructural, Universidade do Algarve, Institute for Biotechnology and Bioengineering, Portugal
SP englisch
PO Schottland
EA pdf-Datei und Poster (Titel: Probing PrP at the cell surface with FRET and FLIM)