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33 Cards in this Set
- Front
- Back
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drug interaction sites by % |
GPCR's 45% |
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number of genes in the human genome
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about 19,000 |
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types of methods for detecting protein-protein interactions |
Molecular biology methods: yeast two hybrid screening |
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For FRET, these two peaks must overlap |
donor fluorescence and acceptor absorption
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FRET stands for |
Fluorescence resonance energy transfer
-or- Forster resonance energy transfer |
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definition of FRET
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nonradiative energy transfer from an excited molecule to another nearby molecule
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formulas for FRET
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E = 1/(1+r/R0) = (# quanta transferred)/(# quanta absorbed by D)
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types of FRET donor-acceptor pairs
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fluorsecent proteins
organic dyes chelates of lanthanides (Eu, TR-FRET) |
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advantage of using organic dyes for FRET pairs
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improved photo and pH stability
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Equation used to relate absorbance to concentration |
Beer-Lambert Law: A = epsilon*b*c
epsilon = molar absorptivity b = path length |
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What does absorbance mean? |
molecules absorb specific wavelengths of light that promote them to excited state; excited state is quantized representing unique configurations of electrons, and vibrational and rotational modes |
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Chemiluminescence definition |
emission of light from a molecule that has previously absorbed light energy from a chemical reaction |
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Stages of fluorescent emission |
1 - excitation - photon absorbed 2 - excited state lifetime 3 - fluorescent emission |
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Stokes shift |
difference between absorption and emission wavelength |
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reason why emission wavelength is higher (from stokes shift) |
energy dissipation during excited state lifetime; lower energy => higher wavelength |
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How to minimize background fluorescence |
Select filters that reduce transmission of E2 relative to E1
Select probes that absorb and emit at longer wavelengths |
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Fluorescence quantum yield definition |
ratio of number of molecules fluorescing to number excited |
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Factors influencing fluorescence quantum yield |
excitation wavelength lifetime of state temperature pH solvent |
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Molecular factors favoring fluorescence |
planar conjugated sterically uncrowded EDG's fused rings rigid chelation to metals |
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fluorescence probes |
chromophores, localize to specific region of specimen and respond to specific stimulus |
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Which is more sensitive for quantification, fluorescence or absorbance spectrometry? |
Fluorescence |
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Chemiluminescence difference from fluorescence spectroscopy |
energy for absorption provided by chemical reaction rather than light
No source or primary filter needed |
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Example of chemiluminescence labeling reagent |
Luminol |
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Methods of two-color FRET |
Sensitized emission
Donor dequenching
Fluorescence lifetime imaging (FLIM)
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Sensitized emission (for FRET) definition |
D excitation/A emission
Issues: spectral bleedthrough contamination |
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Donor dequenching (for FRET) definition |
measuring the intensities of an identical donor fluorophore in the absence and presence of the acceptor |
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FLIM (for FRET) definition |
measuring the reduction in the D lifetime that results from quenching in the presence of an acceptor |
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Limitation of FRET, how to address these |
donor and acceptor are prompt fluorophores with short half-lives
background fluorescence
Solution: TR-FRET (time resolved FRET) |
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TR-FRET basics |
uses long-lived fluorophores (lanthanides)
uses time-resolved detection (delay between excitation and emission detection) |
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Why are lanthanides good for TR-FRET |
very long Stokes shifts
long emission half-lives (usec to msec) |
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Common mechanism of BRET |
Coelenterazine oxidized by luciferise through dioxetane intermediate |
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Example types of cell viability assays and their advantages |
ATP detection assay (most sensitive, least steps, quickest, small interference
Tetrazolium or resazurin reduction (cheap, adequate performace)
Fluorogenic protease (less cytotoxic, multiplexing) |
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Important factors to remember for any cell viability assay |
Use tightly controlled, consistent source of cells
perform appropriate characterization of assay conditions (reagent concentration, incubation time) |
