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

Protocols & Examples

Protocols for transfection of YCs expressed in reticulum

Folding of proteins expressed in endoplasmic reticulum might be problematic, so incubation at 30ºC for some hours is recomended before measurement.

HeLa cells seeding:

  • Seed 15.000 cells per dish (1,5 mL) in Mat-Tek P35G-1.5-14-C dishes for 24 h.

Transfection (procedure for 1 dish):

  • Prepare DNA solution: dilute 3 µL FuGENE in 100 µL Opti-MEM and mix thoroughly, add 1 µg of DNA, mix again and incubate 30 min at room temperature.
  • Wash the cells gently with PBS x 3.
  • Add 1400 µL of Opti-MEM.
  • Add the DNA solution prepared previously.
  • Incubated at 37ºC overnight (8-10 h). Important: Opti-MEM does not contain antibiotic!
  • Remove transfection mixture.
  • Wash gently with PBS x 3.
  • Add DMEM high glucose + 10 % FCS.
  • Allow cells to express YCs proteins for 3-6 days (incubate at 37ºC and change medium as usual).

Measurement:

  • Incubate at 30ºC (4-8h) to allow correct folding.
  • Use medium without phenol red or the corresponding buffer for measurements.

 

 

Fluorescent Dyes

 

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