Calcium imaging

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

Card 2

Front

measures Ca2+uptake (influx+ efflux)

Back

Ca-45 radioactive

Card 3

Front

can measure intracellular levels in living cells

Back

Preview of the back of card 3

Card 4

Front

shift in the wavelenght of light from peak excitation to peak emmision

Back

Preview of the back of card 4

Card 5

Front

1. Single wavelength dyes, 2. ratiometric dyes, 3. protein-based sensors

Back

Preview of the back of card 5
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