Wednesday, April 29, 2015

Product Focus: Cal-520™, Cal-590™ & Cal-630™ Calcium Detection Reagents

Benefits

  • Excellent cellular retention, enabling Ca2+ assays of probenecid-sensitive GPCRs and Ca2+ channels.
  • Robust, significantly imporved S/N ratio than those of Fluo-3 AM, Fluo-4 AM and Rhod-2.
  • High sensitivity and >10 times fluorescence enhancement.
  • Multicolour detection – Long Ex/Em wavelengths of Cal-590™ & Cal-630™ make these dyes compatible for use with green fluorescent protein (GFP) cell lines.

Features

  • Predominantly localised in cytosols.
  • Convenient, can be easily detected by standard filter sets for FITC, TRITC/Cy3 and Texas Red®
  • Versatile packing sizes to meet your special needs.



Cal-520™, Cal-590™ and Cal-630™ provide the most robust homogeneous fluorescence-based assay tools for detecting intracellular calcium mobilisation. They are fluorogenic calcium-sensitive dyes with a significantly improved signal-to-noise ratio and intracellular retention compared to the existing calcium indicators (such as Fluo-3 AM, Fluo-4 AM and Rhod-2 AM). Cells expressing a GPCR or calcium channel of interest that signals through calcium can be preloaded with Cal-520™ AM, Cal-590™ AM or Cal-630™ AM which can cross the cell membrane. Once inside the cell, the lipophilic blocking groups of Cal 520™ AM, Cal-590™ AM or Cal-630™ AM are cleaved by intracellular esterases, resulting in a negatively charged fluorescent dye that stays inside cells. Their fluorescence is greatly enhanced upon binding to calcium. When cells are stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increase the fluorescence of Cal-520™, Cal-590™ or Cal-630™. The characteristics of high sensitivity and >100 times fluorescence enhancement make Cal-520™ AM, Cal-590™ AM or Cal-630™ AM ideal indicators for the measurement of cellular calcium. The high S/N ratio and better intracellular retention make the Cal-520™, Cal-590™ or Cal-630™ calcium assay a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.

Besides their convenient excitation wavelengths and large fluorescence enhancement by calcium, Cal-520™, Cal-590™, and Cal-630™ are predominantly localised in cytosols unlike Rhod-2 and x-Rhod-1 that are mainly localized in mitochondria. In addition, the long Ex/Em wavelengths of Cal-590™ and Cal-630™ make these dyes perfect calcium indicators compatible for multicolour detection with green fluorescent protein (GFP) cell lines. In addition, Cal-520™, Cal-590™ or Cal-630™ calcium assays are optimised to be compatible with most of the existing fluorescence instruments. Cal-520 can be well excited at 488 nm, and used with FITC filter set. Cal-590 is optimized to be excited at 555 nm, and used with TRITC/Cy3 filter set. Cal-590 is optimized to be excited at 594 nm, and used with Texas Red® filter set. The spectral and calcium binding properties are summarized below (see Table 1).

Table 1. Spectral and Ca2+–Binding Properties of Cal-520™, Cal-590™
Ca2+ Indicator Excitation (nm) Emission (nm) Kd (nm) Filter Set
Cal-520™ 492 514 320 FITC
Cal-590™ 573 588 561 TRITC/Cy3
Cal-630™ 608 626 792 Texas Red®    

Product Range

  • Cal-520™, AM Cell-Permeable
  • Cal-520™ Sodium Salt
  • Cal-520™ Potassium Salt
  • Cal-520FF™, AM cell-permeable
  • Cal-520FF™ Potassium Salt
  • Cal-520-Dextran Conjugate MW 3,000
  • Cal-520-Dextran Conjugate MW 10,000
  • Cal-590™ AM Cell-permeable
  • Cal-590™ Sodium salt
  • Cal-590™ Potassium Salt
  • Cal-630™ AM Cell-permeable
  • Cal-630™ sodium salt
  • Cal-630™ potassium salt



Because of the importance of Ca2+ in biology, numerous techniques/methods for analysing the mechanisms of cellular and/or subcellular Ca2+ activity have been established.  Although each method for analysing Ca2+ activity has certain advantages of the others, each also suffers for drawbacks.  With the outstanding properties of Cal-520™, Cal-590™ & Cal-630™, calcium detection reagents provide a new powerful tool for intracellular calcium analysis and monitoring in a variety of biological systems.

The interests of many researchers in Ca2+ analysis has shifted from the cellular level to the subcellular level.  It has been found that Ca2+ is not even distributed throughout the whole cell and that intracellular heterogeneity of Ca2+ (such as Ca2+ waves and Ca2+ sparks) is observed in a variety of cells (e.g. oocyte, heart muscle cell, hepatocyte, and exocrine cells).  With the advent of the confocal laser scanning microscope (CLSM) in the 1980’s and advanced miscroplate readers in 2000’s (such as FLIPR, FDSS and NOVOStar dedicated for intracellular Ca2+ detections), the measurement of intracellular Ca2+ has accelerated significantly.  Confocal laser scanning microscopy and more recently multiphoton microscopy allow the precise spatial and temporal analysis of intracellular Ca2+ signalling at the subcellular level in addition o the measurement of its concentration.



Where can I find more information about AAT Bioquest?

Visit the manufacturer page at www.stratech.co.uk/aatbioquest, email info@stratech.co.uk or call +44 (0) 1638 782600.

Stratech Scientific is a distributor of high quality, competitively priced, reliable products for research laboratories throughout the UK and Europe. Please contact us to find out which ranges we can supply in your country.
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