Seppro rubisco spin columns

Two grams of leaf tissue from A. thaliana (Columbia-0) at 21 days after germination was used for cell fractionation as described previously (20 (link), 22 (link), 26 (link), 27 (link)). Briefly, fresh tissues were ground using a Polytron homogenizer (Brinkmann Instruments) under ice-cold microsome isolation buffer solution [50 mM Hepes/KOH (pH 7.5), 250 mM sorbitol, 50 mM KOAc, 2 mM Mg(OAc)₂, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 2 mM phenylmethylsulfonyl fluoride, and 1% (v/v) protease inhibitor cocktail (160 mg/ml benzamidine-HCl, 100 mg/ml leupeptin, 12 mg/ml phenanthroline, 0.1 mg/ml aprotinin, and 0.1 mg/ml pepstatin A)]. The homogenate was filtered with four layers of cheesecloth to remove debris and enriched by centrifugation at 1000g on a Beckman Avanti 30 (Beckman Coulter Life Sciences) for 10 min at 4°C. The supernatant was obtained by ultracentrifugation at 200,000g for 20 min at 4°C using a Beckman Optima Ultracentrifuge (Beckman Coulter Life Sciences). The volume of the soluble fraction was concentrated to 1 ml using an Amicon Ultra-4 10K Centrifugal Filter (Millipore). Seppro Rubisco spin columns (Sigma-Aldrich) were used for the removal of RuBisCO from the concentrated samples.