Cells (108) were washed in PBS and resuspended in SoTE (0.6 M sorbitol, 2 mM EDTA, 20 mM Tris-HCl, pH 7.5) (73 (link)). Cells were incubated with 0 or 0.16 mg of digitonin per mg of protein for 4 min at 25°C before centrifugation at 14,000 × g for 2 min. The supernatants containing both the cytosolic and glycosomal proteins were analyzed by blue native PAGE (BN-PAGE) on a precast (3 to 12%) Bis-Tris polyacrylamide gel (Invitrogen) according to standard methods.
Corresponding Organization : Centre de Résonance Magnétique des Systèmes Biologiques
Other organizations :
Institut National des Sciences Appliquées de Toulouse, Inserm, Université Grenoble Alpes, Nagasaki University, The University of Tokyo, Université de Toulouse, Université Toulouse III - Paul Sabatier
Digitonin concentration (0 or 0.16 mg per mg of protein)
dependent variables
Cytosolic and glycosomal protein profile analyzed by blue native PAGE (BN-PAGE)
control variables
Cell number (10^8 cells)
Washing in PBS
Resuspension in SoTE buffer (0.6 M sorbitol, 2 mM EDTA, 20 mM Tris-HCl, pH 7.5)
Incubation time (4 min)
Incubation temperature (25°C)
Centrifugation speed (14,000 × g)
Centrifugation time (2 min)
controls
Positive control: Cells incubated with 0.16 mg of digitonin per mg of protein
Negative control: Cells incubated with 0 mg of digitonin per mg of protein
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