Dutch Boltz - Molecular Devices

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A Dual-Laser Configuration on a FLIPR 384Instrument In order to utilize the FRET based voltage sensor probes for ion channel screening on FLIPR a 409nm krypton laser was introduced onto the instrument. At left, the krypton-argon laser substitution diagram for the primary argon laser. The 409nm beam was introduced co-linear to the 488nm argon laser path by the addition of beam steering optics. Two dichroic filters are required to pass the krypton light beam through a mirror and into the FLIPR optical detection path. The argon light beam passes through the dichroic filter and mirror into the FLIPR optical detection path. At right, a photograph of the 409nm krypton laser beam path through the beam steering optics and into the FLIPR. The substitution of the primary light source in conjunction with the use of multiple filter sets allows one to use a wide range of distinct fluorescent probes. 6/14/2004 Cardiovascular Diseases 24
Coumarin-Donor Oxonol-Acceptor Fluorescent Dye Components of the FRET Membrane Potential Assay A two dye configuration is used in the FRET based membrane potential assay. At left, the coumarin labeled phospholipid CC2-DMPE molecule which partitions itself into the outer leaflet of the plasma membrane and serves as the FRET donor. Negative charges from the coumarin and phosphate groups prevent the CC2-DMPE donor molecule from crossing the phospholipid bilayer. At right, the negatively charged bis-(1,3-dialkylthiobarbituric acid) trimethane oxonol acceptor molecule whose position within the bilayer is determined by the membrane potential of the cell. Both components are very bright fluorophores with a large spectral separation between emissions making signal separation and detection feasible. Coumarin excitation is at 410nm and emission at 460nm as compared to Oxonol with an excitation at 540nm and emission at 570nm. 6/14/2004 Cardiovascular Diseases 25
Page 1 and 2: Dutch Boltz • Dutch has been at M
Page 3 and 4: Today’s s Presentation • Review
Page 5 and 6: Voltage Dependent FRET Mechanism of
Page 7 and 8: Comparison of the Two “I” Plate
Page 9 and 10: Bleed Down of “FR IPR” Membrane
Page 11 and 12: Components Used To Modify FLIPR to
Page 13 and 14: FRET Dye Protocol • 30,000 HEK-2
Page 15 and 16: 384 Well FLIPR Ratio Traces for a 1
Page 17 and 18: Inhibition of Membrane Depolarizati
Page 19 and 20: Areas for Improvement • Can Optic
Page 21 and 22: For More Details and Insight See Ri
Page 23: Abstract Ion channels are an attrac

Dutch Boltz - Molecular Devices

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