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studies/ColdChloroEnvelope/protocols/Enrichment of envelope membranes.md

+Enrichment of envelope membranes was carried out according to a given protocol (Ferro et al., 2003) with modifications. The intact chloroplast fraction was vigorously resuspended in 2 mL of buffer medium (10 mM MOPS-NaOH, pH 7.8) and kept on ice for 10 min to allow osmotic disruption of chloroplasts. To prevent protease-driven protein degradation the buffer medium was complemented with a protease inhibitor cocktail (cOmplete, EDTA-free, Sigma Aldrich; www.sigmaaldrich.com). At this step, 100-μL samples were collected from the lysate for the MS-based identification of total chloroplast proteins.
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+A three-step Suc gradient (bottom to top: 4 mL 0.93 M, 0.6 M, and 0.3 M Suc) prepared in Ultra-Clear tubes (16 × 102 mm, Beckman Coulter; www.beckmann.de) was overlaid with 1 mL of disrupted chloroplast preparation. After ultra-centrifugation (70,000g for 1 h at 4°C, with no brake, on a swing-out rotor SureSpin 630, Thermo Fisher Scientific; www.thermo-fisher.com) the yellowish envelope fraction was collected from the interphase between 0.93 M and 0.6 M Suc (Supplemental Fig. S1). This fraction was 2× diluted with double distilled water and the envelope membranes were collected by ultra-centrifugation (400,000g for 20 min at 4°C on a ST120AT rotor, Thermo Fisher Scientific; www.thermofisher.com,). The resulting membrane fraction was resuspended in 1 mL double distilled water to remove any remaining Suc. To remove membrane-associated proteins the envelope membranes were resuspended in 1 M of sodium carbonate (Na2CO3) medium and centrifuged again, as described above. This washing step was repeated five times.
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studies/ColdChloroEnvelope/protocols/Isolation of Chloroplast Envelope Membranes.md

+Isolation of Chloroplast Envelope Membranes
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+The envelope membrane isolation procedure can be divided into two steps: (1) isolation of intact chloroplasts and (2) enrichment of envelope membranes from these chloroplasts using a Suc step gradient (Supplemental Fig. S1). The isolation of intact chloroplasts was carried out according to an existing protocol (Kunst, 1998) with some modifications: 200 g leaf material was chopped off 34-d-old Arabidopsis plants (cold acclimated and control plants kept at 22°C) and transferred to ice-cold homogenization buffer medium (0.45 M sorbitol, 20 mM Tricine-KOH, pH 8.4, 10 mM EDTA, 10 mM NaHCO3, and 0.1% [w/v] fatty-acid free bovine serum albumin). The ratio of buffer volume to weight of leaf material was 3:1 (v/w). In a glass beaker the buffer/leaf mixture was further cooled in ice water to limit metabolic activity to a minimum. After 30 min the mixture was transferred to a 1 L stainless steel beaker. For a controlled rupture of the leaves, the blender was successively run for 1 s at low, 1 s at medium, and 1 s at high settings (Waring commercial heavy-duty blender). This procedure was repeated twice. The disrupted leaf material was than filtered through three layers of Miracloth (http://www.merckmillipore.com), placed in a funnel, and the flow through collected in an ice-cooled Erlenmeyer flask. From this suspension the chloroplast fraction was collected by centrifugation (1,000g for 10 min at 4°C) and gently resuspended in 8 mL resuspension buffer medium (0.3 M sorbitol, 20 mM Tricine-KOH, pH 7.6, 5 mM MgCl2, and 2.5 mM EDTA) using a natural bristle paint brush.
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+A Percoll gradient was prepared by mixing equal volumes of 2× concentrated resuspension buffer medium and pure Percoll. Of this mixture, 30 mL was transferred to 36-mL centrifuge tubes and centrifuged (Sorval SS34 fixed-angle rotor) at 43,400g for 30 min at 4°C with no brake. Two Percoll gradients were enough for 200 g of leaf material. The Percoll gradient was overlaid with the resuspended chloroplast suspension. After centrifugation in a HB4 swing-out rotor (13,300g for 15 min at 4°C, with no brake), two distinct green bands appeared (Supplemental Fig. S1). The upper band, containing broken chloroplasts, was removed using a water jet pump and the lower band was collected using a wide-open Pasteur pipette. This fraction was transferred to a SS34 tube and diluted with 3 volumes of resuspension buffer medium. From that suspension, intact chloroplasts were collected by centrifugation (HB4 rotor, 2,700g, 5 min, no brake).
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