OrganellarMembraneIsolation.md

2.5 Isolation of organellar membranes

Membranes were isolated from the collected organellar fractions (F1-4) as described by Fujiki et al. (1982) and Reumann et al. (1995) with modifications. The obtained fractions were slowly diluted with hypo-osmotic buffer (20 mM HEPES-KOH, pH 6.8 and 0.8 mM MgCl2) and incubated on ice for 30 minutes to osmotically disrupt the organelles. The suspension was subjected to 10 freeze/thaw cycles (freezing in liquid nitrogen, thawing at room temperature) to lyse efficiently the organelles. After each thaw cycle the sample was homogenized by mixing. Membranes were sedimented by centrifugation at 100,000 g at 4°C for 1 h and washed in 100 mM sodium carbonate (pH 11.5) to remove membrane- associated proteins. A second centrifugation step (100,000 g at 4°C for 1 h) was performed to harvest the organellar membranes. The membrane pellet was resuspended in 20 mM HEPES-KOH, pH 6.8 and 0.8 mM MgCl2 and stored at -80°C for further experiments. Concentration of the membrane proteins were determined using Pierce BCA protein assay kit (ThermoFisher Scientific).

• Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982). Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J. Cell Biol. 93, 97–102. doi: 10.1083/jcb.93.1.97
• Reumann, S., Maier, E., Benz, R., and Heldt, H. W. (1995). The membrane of leaf peroxisomes contains a porin-like channel. J. Biol. Chem. 270, 17559–17565. doi: 10.1074/jbc.270.29.17559