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).
- ATCC HeLa cells grown in DMEM high glucose.
- Glass Bottom Culture Dish (Mat-Tek
cDNA for expression of YCs
All constructions were kindly provided by Roger
- 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)
Calcium measurements should be performed in medium/buffer without Phenol
Red (it can interfere in fluorescence signal).