In:
Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2014, No. 7 ( 2014-07), p. pdb.prot073270-
Kurzfassung:
Fluorescent Ca 2+ indicators are widely used to measure the concentration of free Ca 2+ ([Ca 2+ ] free ) in biological processes. By calibrating the dye under the same experimental conditions as employed during its planned use, the actual [Ca 2+ ] can be calculated from the measured fluorescence. When using non ratiometric dyes, such as the Oregon Green BAPTA (OGB) family of dyes or the Fluo dyes, the steady-state affinity ( K d ) and the ratio between the maximal and minimal fluorescence ( F ratio = F max / F min ) of the particular dye are needed for this conversion. Although these values are usually given by the manufacturer, we consistently find that the actual values can differ between various batches delivered by the companies that make the dyes. In this protocol, we provide the recipe for a series of solutions with a known and tightly buffered [Ca 2+ ] free and describe how to use these mixtures to determine the exact K d and F ratio of a fluorescent Ca 2+ dye.
Materialart:
Online-Ressource
ISSN:
1940-3402
,
1559-6095
DOI:
10.1101/pdb.prot073270
Sprache:
Englisch
Verlag:
Cold Spring Harbor Laboratory
Publikationsdatum:
2014
