Arabidopsis mitochondrial protein preparation

Protocol from Isolation of intact, functional mitochondria from the model plant Arabidopsis thaliana. Lee J Sweetlove 1, Nicolas L Taylor, Christopher J Leaver. Methods Mol Biol. 2007:372:125-36.doi: 10.1007/978-1-59745-365-3_9.

Procedure:

Three-week-old WT plants were fully homogenized in isolation buffer (0.3 M sucrose, 5 mM tetrasodium pyrophosphate, 10 mM KH₂PO₄, pH 7.5, 2 mM EDTA, 1% PVP40, 1% BSA, 5 mM cysteine and 20 mM ascorbic acid), filtered through Miracloth and then centrifuged at 5000 × g for 10 min at 4℃. The supernatant was collected and centrifugation at 20,000 × g for 10 min at 4℃. The pellet containing crud mitochondria was resuspended in buffer with 0.3 M sucrose, 1 mM EGTA, and 10 mM MOPS/KOH, pH 7.2 and further centrifuged through a Percoll density gradient consisting of 18%, 25%, and 50% Percoll solution at 40,000 × g for 55 min at 4℃. Intact mitochondria at interface of 25-50% Percoll was collected. Protein concentration was determined using a DC Protein Assay kit (BioRad, 5000116). The mitochondrial protein was solubilized in 2×sample buffer.