Calcium imaging 0.0 / 5 ? BiologyGeneticsUniversityNone Created by: molgCreated on: 18-05-18 12:44 Colourmetric assay free Ca2+ in blood, urine, cell lysates- no rate/flux/can't localise 1 of 17 Ca-45 radioactive measures Ca2+uptake (influx+ efflux) 2 of 17 Fluorescent Ca indicatiors can measure intracellular levels in living cells 3 of 17 Stokes shift shift in the wavelenght of light from peak excitation to peak emmision 4 of 17 Different types of fluorescence dyes 1. Single wavelength dyes, 2. ratiometric dyes, 3. protein-based sensors 5 of 17 Single wavelength dyes BAPTA- calcium chelator/ Fluoroisothiocynate (FITC) (excitation- blue, emission= green) 6 of 17 mGlu1R produces a single Ca2+ spike then desensitises 7 of 17 mGlu5R oscillatory response 8 of 17 ratiometric dyes 2-excitation or emission wavelengths 9 of 17 FURA-2 2 excitation peaks- bound=340nm/ unbound=380nm 10 of 17 Isobestic point where the 2 spectra meet- peak values need to be either side of this point 11 of 17 INDO-1 2-emmission wavelengths 12 of 17 Genetically-encoded indicators directed too selected populations of cells/ used to generate transgenic animals/expression can be controlled 13 of 17 Cameleons consists of; calmodulin (ca binding protein), MI3 (CaM binding protein)- connect 2 fluorescent proteins 14 of 17 FRET- fluorescence Resonance Energy Transfer on ca2+ finding a conformational change brings the B/GFPs together to induce FRET 15 of 17 Inverse Square Law Intensity = 1/distance squared- FRET only occurs when donor and recipient proteins are close together 16 of 17 GCamPs GFP, calmodulin and M13- GFP fluorescences when ca2+ binds- Much brighter than FRET 17 of 17
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