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Cross-contamination of cytosolic proteins into the membrane fraction is typically <10%.
According to Figure 1a, the proportion of PDE3B in the plasma membrane and internal membrane fraction is 1∶5.
Most of the ER, vacuolar membranes and plasma membrane are found in the heavy membrane fraction, whereas the light membrane fraction is enriched in Golgi membranes [24].
This membrane fraction is used as the source of GCS enzyme in this assay, as purified fungal GCS is not yet available.
Anti-alpha tubulin antibody in Western blotting is a useful marker to check whether the plasma membrane fraction is contaminated with cytosolic proteins because cytoskeletal proteins are the most common contaminants during plasma membrane extraction.
It has been previously reported that most of the ER, vacuolar membranes and plasma membrane are found in the heavy membrane fraction, whereas the light membrane fraction is enriched in Golgi membranes [24].
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For further outer-membrane fractionation, the pellet (total membrane fraction) was resuspended sterilized distilled water.
Here, the E. coli membrane fraction was obtained through ultracentrifugation method.
A cell membrane fraction was prepared according to the previously described method (Frieman and Cormack 2004; Hara et al. 2012a).
The crude membrane fraction was collected from the gradient interface.
Postnuclear supernatants were further ultracentrifuged, and the pellets (membrane fraction) were resuspended in lysis buffer.
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