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Proteins were extracted using the native extraction protocol.
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The resulting chloroplast pellet was used for native peptide extraction.
Membrane and soluble protein fractions were obtained using the Proteoextract Native Membrane extraction kit (Calbiochem).
Native protein extraction resulted in low yields of Exo1 in cdc13-1 mutants grown at 36°C for more than 3 h.
Membrane and cytoplasm fractions were separated with a native membrane extraction kit (Merck Millipore) according to the manufacturer's protocol.
As this protocol is based on a native protein extraction, it identified - in relative terms - the lowest number of membrane proteins.
For native protein extraction, the pellet was snap frozen then resuspended in 35 µl solubilization buffer (50 mM NaCl, 50 mM imidazole, 2 mM 6-aminohexanoic acid, 1 mM EDTA, pH 7), and solubilized with 8 µl digitonin (20% stock in water).
For this comparative study we selected an in gel digest method adapted from, conducted in combination with an SDS-based and native protein extraction, two filter-aided (FASP (Filter Aided Sample Preparation) adapted from and a recent enhancement termed eFASP adapted from, and two in solution procedures (RapiGest, adapted from, and RapidACN adapted from ).
In this study, membrane protein was extracted using a Native Membrane Protein Extraction Kit (Calbiochem, Cambridge, MA, USA) according to the manufacturer's instructions.
Membrane and cytoplasmic proteins from ES-2 cells were extracted with ProteoExtract® Native Membrane Protein Extraction Kit (Calbiochem) according to the manufacturer's instructions.
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