In this video, we demonstrate the introduction of bacterial effector proteins into mammalian cells by electroporation. The intracellular localization of the bacterial effector proteins is detected by confocal microscopy following staining with specific fluorophore-conjugated antibodies.
Protocol
1. Electroporation Place 400 µl of cell suspension in a pre-chilled cuvette and add 20 µg of selected protein to the cuvette (50 µg/ml). Flick the cuvette gently ~10 times to mix without damaging cells. Note: The cuvette may also be inverted several times to mix thoroughly, but do not pipet up and down or vortex to avoid damaging cells. Dry the outside of the cuvette with a paper towel or other wiper to avoid electrical arcing in the electro…
Declarações
The authors have nothing to disclose.
Materials
0.25% Trypsin-EDTA Solution
Cellgro
25-050-Cl
0.4cm Gap-disposable electroporation cuvettes
Bio-Rad
165-2088
100% Methanol
Any
N/A
Flammable, Toxic
Bovine Serum Albumin (BSA)
Sigma-Aldrich
A4919
Cell Counting Apparatus – Hemocytometer or Coulter Counter