Fluorescent Proteins:
Introduction - Quantifying calcium - Definitions - Protocols & Examples
 

Protocols & Examples

Fluorescent Proteins - Material and solutions

Material and solutions are detailed below. Changes should be made to adapt them to your own needs (see some tips here).

Cells seeding:

  • ATCC HeLa cells grown in DMEM high glucose.
  • Glass Bottom Culture Dish (Mat-Tek P35G-1.5-14-C).

cDNA for expression of YCs

All constructions were kindly provided by Roger Y. Tsien.

  • YC2: shows a biphasic Ca2+ response (suitable for [Ca2+] between 100 nM and 10 µM)
  • YC3.1: shows a monophasic Ca2+ response (suitable for [Ca2+] between 1 nM and 100 µM)
  • YC3.1er: equivalent to YC3.1 but it has the calreticulin leading sequence in the N terminus and the retention signal KDEL in the C terminus. This YC is retained in the ER.
  • YC4erQ69K: low affinity indicator (suitable for 10 -1000 µM Ca2+). It has the calreticulin leading sequence in the N terminus and the retention signal KDEL in the C terminus, so it is also retained in the ER.

Solutions and reagents

  • FuGENE (Roche Diagnostics)
  • Opti-MEM (Gibco-BRL)
  • cDNA in ddH2O
  • DMEM high glucose (with and without Phenol Red)
  • PBS

Calcium measurements should be performed in medium/buffer without Phenol Red (it can interfere in fluorescence signal).

 

Fluorescent Dyes

 

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