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The pipette is angled so the body of the pipette contacts the upper lip of the tube. Cover the Percoll gradients with aluminum foil, place in test tube rack embedded in an ice bucket with ice slush and store in the cold room until use. Sterile filter into sterile mL storage bottle. Following three 5-min washes in sterile water, seeds are placed on water-moistened Whatman 1 filter paper disc in petri dishes. Plants are watered daily and fertilized weekly with a 0.

Plants with three to four true leaves are used for chloroplast isolation. Twenty-seven hours prior to the chloroplast isolation, tomato plants are transferred to the dark to reduce starch. Starch-filled chloroplasts lyse and reduce yields of intact chloroplasts. Solutions are autoclaved or sterile filtered. If chloroplasts are to be used for proteomics studies, all steps should be performed wearing latex gloves.

All steps of the protocol are carried out quickly in the cold room. An ice slush is used for chilling and transporting centrifuge bottles and tubes within ice buckets. This protocol is scaled for performing three g leaf chloroplast preparations simultaneously. To develop our chloroplast isolation protocols, we considered the published protocols for isolating tomato leaf chloroplasts for proteomics [ 52 ], protein import and biochemistry [ 53 , 54 ], and chloroplast genome isolation [ 55 — 57 ], as well as methods for plastid isolation from tomato fruit [ 48 — 50 , 58 ] Additional file 1 : Table S1.

While protoplasts clearly provide the highest chloroplast yields [ 59 ], the lengthy preparation times and use of cell wall hydrolyzing enzymes are disadvantageous to proteomics studies studying biotic stress, as this method generates cell wall-derived elicitors that could trigger plant-defense responses Table 1.

Our protocol primarily builds upon the Arabidopsis chloroplast isolation methods and stromal protein isolation methods of van Wijk et al. Figure 1 provides a flow chart that summarizes the basic sequence of events for: 1 the isolation of tomato chloroplasts, 2 isolation of chloroplast stromal proteins, and 3 proteomics sample processing and data analysis. In addition to the detailed protocols below, we provide a streamlined workflow checklist for chloroplast isolation that can easily be used for tracking steps Additional file 2 : Table S2.

Leaf segments were added in two batches into the blender. Flowchart of events for chloroplast isolation, stroma extractions and proteomics data generation and analyses. The 1X Grinding Buffer:blender cup volumes and the brand and settings of the blender used are critical parameters. The configuration of the blades and rotations per minute of the blades for each blender will be different. The blender settings are increased incrementally until the blades rapidly mix the 1X Grinding Buffer ice slush and leaves within the blender cup.

If the setting is too low, inadequate homogenization will occur e. If the setting is too high, chloroplasts will be sheared, the homogenate will be dark green and foamy and yields will be significantly reduced. In an optimal homogenization protocol, the homogenate is pale green and a substantial amount of leaf debris remains and is captured by the Nitex filter.

While longer homogenization times decrease the amount of leaf debris, chloroplast yields decline due to lysis of intact chloroplasts and the percentage of intact chloroplasts was typically Using short, optimized blending times, intact chloroplast yields increased 3. Additional parameters critical for high yields include: leaf age and texture, the tissue:buffer ratio, the amount of the ice to liquid ratio of the 1X Grinding Buffer.

Similar to the Arabidopsis protocol [ 42 ] and unlike other tomato plastid preparations Additional file 1 : Table S1 , we used a tissue to buffer ratio to assure adequate buffering capacity and movement of tissue in the homogenization process. We also discovered that adding leaves in two sequential 7. The optimization of these steps increased yields to 4. Transfer dark-treated tomato plants into a room without direct light.

Excise young leaves with a razor blade; thick and dark-green older leaves and damaged leaves of any age are not used. Using a razor blade and a glass plate as a cutting surface, remove the midrib from each leaflet; this step is critical for efficient homogenization. To minimize tearing of leaves, change razor blades frequently to assure a clean cut. All steps are performed as quickly as possible in the cold room. Fit three wide-mouthed funnels with two pieces of Nitex pre-wetted with water and place funnel in each 1-L flask.

Homogenize leaves in small batches to assure the optimal amount of shear to release but not damage chloroplasts. Add approximately 7. Add the remaining 7. If tissue is limiting, the brei can be returned to the blender cup and homogenized with 50 mL 1 X GB ice slush for 2 s to increase yields. Pour the homogenate through two layers of Nitex and collect in the 1-L flask.

Repeat the homogenization procedure with additional 7. Homogenates for each treatment are pooled and allowed to passively filter through Nitex. Meanwhile, rinse the blender cup with water and dry. Process the additional g leaf preparations immediately. Gently squeeze the Nitex to recover residual buffer from the leaf debris. Approximately — mL of homogenate will be recovered per g leaf preparation.

Pour approximately mL of each homogenate into a prechilled, mL centrifuge bottle 1 bottle per g preparation. Balance bottles with 1 X GB as needed; use the extra centrifuge bottle as a balance. Immediately pour off each light-green supernatant into its own mL beaker and mark location of pellet.

Add the remaining homogenate to its corresponding crude chloroplast pellet. Balance bottles and centrifuge as described above. Pour the supernatant for each g preparation into its corresponding mL beaker. The green pellets contain both intact and broken chloroplasts and other cellular components. Faint white starch rings will be observed at the bottom of the bottle. Use a paintbrush that is pre-wetted in cold 1X GB to gently resuspend the pellet until no clumps remain.

At this point, the crude chloroplast suspension should be thin enough to drip off the tip of the paintbrush. Add an additional 0. This dilutes the suspension, prevents chloroplast aggregation and facilitates sample loading onto the Percoll gradients, as well as assuring better separation of intact and broken chloroplasts.

Use two Percoll gradients for one g chloroplast preparation to avoid over-loading the gradients. Use a wide-bore 1-mL tip and Pipetman P to gently overlay half of the crude chloroplast suspension onto a Percoll step-gradient Fig.

The gradient is placed in a test-tube rack immersed in an ice slush bath. Repeat for additional samples. Transport the gradients in the ice bath to the high-speed centrifuge. Wipe the condensation off the tubes, place the tubes in prechilled mL tube adaptors and rubber sleeves within the JS5. The rotor is stopped with the brake. Carefully transfer the gradients to the test-tube rack in the ice bucket.

Isolation of tomato chloroplasts on Percoll gradients. Place a Percoll gradient in a rack on the cold room benchtop. Do not disrupt the starch pellet at the bottom of the tube. Repeat for the remaining gradients. To the six mL tubes two tubes per g preparation , add 10 to 20 volumes of chilled 1X HS buffer approximately 20 mL.

Seal the tube with parafilm and mix gently by inversion. Remove parafilm and place centrifuge tubes in the JS 5. Return the tubes to the cold room and carefully pour off the supernatant into a mL beaker and discard. Combine the two pellets from the same g chloroplast preparation in a single 2-mL Axygen microfuge tube. Adjust the volume of chloroplasts to 2 mL with cold 1X HS buffer, mix gently by pipetting up and down.

Typical chloroplast yields are This protocol is based on the van Wijk et al. First, the MgCl 2 concentration was reduced from 5. Second, a different proteinase inhibitor cocktail was used. The scheme for extraction of chloroplast stromal proteins is outlined in Fig. Typical yields of stromal protein were 7. Chill rotors, adaptors and centrifuge tubes One day prior to stroma extraction. Add 2 mL of sterile deionized water to the Amicon Ultra 3 K filter reservoir.

The last steps of Amicon filtration unit preparation are described in the protocol below. Immediately prior to use, add the thawed protease inhibitor cocktail. Store on ice until use. Using a P, carefully remove and discard the supernatant. Incubate the chloroplasts for 60 min on ice allowing the chloroplasts to swell and burst.

Transfer the suspension to a 2-mL Tenbroeck tissue grinder. Apply three strokes with intermittent rotation of the piston. Transfer the suspension to a 2-mL Axygen microfuge tube. Repeat with additional chloroplast preparations. Rinse the Tenbroeck tissue grinder between different chloroplast preparations.

Transfer the supernatant stroma to a 3. Mark the lip of each tube with a marker and orient mark towards the outer rim of the rotor to facilitate locating the minute membrane pellet after ultra-centrifugation. Balance the ultracentrifuge tubes using chloroplast lysis buffer if needed.

Complete preparation of the Amicon Ultra 3 K filtration unit. Transfer the water in the chamber using a P to a waste container mL beaker. Reassemble filtration unit and immediately load sample as directed below. Do not allow the filters to dry out. Using a P, remove the supernatant the stroma avoiding the membrane pellet. Transfer stroma to a 1.

Proteins from total leaf extracts, intact chloroplasts, and stromal and non-soluble chloroplast proteins were isolated using the methods described in [ 61 ]. For immunoblots, proteins were transferred to Whatman Protran BA85 nitrocellulose membranes. Membranes were incubated with the secondary antiserum for 1 h, and washed three times in TTBS; chloroplast marker-protein blots were then washed once with 1X TBS for 5 min.

The OEC23 antiserum was raised against recombinant pea OE23 recovered from inclusion bodies Kenneth Cline, personal communication used in [ 65 ]. All antisera were provided by Kenneth Cline U Florida. Membranes were washed three times with PBS. Blots were incubated for 1 h with primary antisera dilution and subsequently washed with three times with PBS. Chlorophyll was extracted from samples using acetone and concentrations were determined as by Lichtenthaler [ 67 ]. The number of intact and ruptured chloroplasts in a field were counted and the percentage of intact chloroplasts calculated.

The gel section containing the most abundant proteins 50—70 kDa was excised and discarded. A MudPIT approach was employed to analyze the trypsin-treated samples and details are provided in [ 70 ]. The first dimension LC fractionation used 20 mM ammonium formate pH 10 and acetonitrile.

The second dimension nanoUPLC method was described previously [ 70 ]. Orbitrap mass analyzer was used for the MS1 scan. For the MS2 scan, the Ion-Trap mass analyzer was used in a rapid scan mode. Only precursor ions with intensity 10, or higher were selected for MS2 scan. Sequence of individual MS2 scanning was from most-intense to least-intense precursor ions. The raw MS files were processed and analyzed using Proteome Discoverer version 2.

The search parameters were the following: trypsin with two missed cleavages, minimal peptide length of six amino acids, MS1 mass tolerance 20 ppm, MS2 mass tolerance 0. Proteins were identified using the deduced tomato proteome [ 71 ] based on the criteria detailed in Bhattacharya et al. Five independent protein localization algorithms ChloroP, TargetP, Predotar, WolfPSort, and YLoc were used to assemble a tomato plastid protein dataset Atlas [ 72 — 76 ], which was used to predict subcellular localization of tomato proteins [ 68 ].

Proteins that reproducibly co-isolated with tomato chloroplast stromal proteins were identified. Relative protein abundance was calculated based on normalized spectral abundance factors NSAF [ 82 , 83 ]. The spectral abundance factor SAF was calculated for each protein.

Numerous methods for isolation of intact plastids and sub-fractionation of chloroplast compartments for proteomics studies in Arabidopsis populate our literature today [ 42 , 46 , 84 ]. In addition, robust methods have been developed for isolation of metabolomics- and proteomics-grade chromoplasts of tomato fruit [ 47 — 51 , 58 ] Additional file 1 : Table S1.

While protocols for isolating chloroplasts for DNA isolation, enzymatic assays and protein import assays have been described [ 53 — 57 , 85 ], rather surprisingly, few chloroplast large-scale proteomics studies have been reported for tomato leaves [ 52 ] Additional file 1 : Table S1.

Therefore, a high-yield method for intact chloroplast isolation and methods for recovery of the membrane and stromal fractions of chloroplasts for proteomics analyses for tomato was needed. Our protocol for chloroplast isolation builds upon methods developed for Arabidopsis chloroplasts [ 42 ] and incorporates many recommendations from foundational studies in spinach and pea [ 86 , 87 ].

Our methods for proteomics-grade tomato chloroplasts are more similar to those used by van Wijk et al. However, unlike the Arabidopsis protocol, our tissue grinding buffer: 1 did not use the anti-oxidant cysteine; 2 included 1 mM MgCl 2 and 1 mM MnCl 2 similar to several tomato protocols, and 3 included fivefold higher BSA 0.

The general scheme for chloroplast isolation, stroma extraction and proteomics processing and data analysis are provided in Fig. Briefly, the deveined tomato leaf homogenate is centrifuged; the pellet contains both intact and broken chloroplasts. It is clear based on the size of the bands at the two interfaces Fig. In the process of refining our chloroplast stroma isolation method, we discovered several parameters that markedly increased tomato chloroplast yield. First, only young, undamaged leaves from 4- to 5-week old tomato plants are used.

These leaves are tender and many are expanding and therefore provide best yields. Second, several parts of the homogenization protocol are critical for high-yields and intact chloroplasts. We found that the slurry status of the 1X Grinding Buffer 1 part ice: 1 part liquid enhanced the yield of intact chloroplasts Table 1. If the Grinding Buffer is too watery, there is excess chloroplast breakage and stroma is lost; if the buffer is too icy, there is insufficient cell breakage and chloroplasts are not released.

Third, the volume of tissue and buffer relative to the blender cup size is critical. If the blender cup is too full, insufficient homogenization occurs; not full enough, foaming protein denaturation and excess plastid breakage occurs.

Fourth, as well established in the literature [ 87 ], the duration of the blender pulse is critical and empirical determination of the optimal blender settings are essential. We found that two 2-sec blender pulses released tomato cell content and retained chloroplast integrity.

Another unique feature of our method is that additional leaf tissue was added after the first 2-sec pulse. Unlike the van Wijk et al. Finally, we also found that by decreasing MgCl 2 in the chloroplast lysis buffer from 5 mM [ 42 ] to 2. With these modifications, typical chloroplast protein yields from tomato leaves yielded 0. Proteins ranging from over kDa to under 20 kDa were resolved indicating the high quality of proteins recovered at different stages in the tomato leaf chloroplast protocol.

The purified chloroplast extracts were enriched for a subset of the proteins in the total leaf extracts Fig. Furthermore, a majority of the abundant proteins found in intact chloroplasts were also present in the non-soluble, membrane fraction after chloroplast lysis. In contrast, the stromal protein fraction is distinct with a small number of superabundant proteins in the to kDa range.

Silver-stained SDS—polyacrylamide gels and immunoblots with protein fractions from the chloroplast stroma isolation protocol. Masses of molecular weight markers are shown in kDa. The RPS6 antisera cross-reacts with several tomato proteins. The kDa RPS6 protein is solely found the total leaf homogenate; several of its cross-reacting proteins are enriched during the steps used for chloroplast stromal protein isolation. The mass kDa of each protein is shown. To evaluate the efficacy of our chloroplast and stroma isolation methods, we determined the levels of three proteins that are known to reside in the chloroplast stroma heat shock protein 70; HSP70 , lumen oxygen evolving complex 23; OEC23 , and thylakoid membranes light harvesting complex proteins; LHCP , as well as one cytosolic protein ribosomal protein S6; RPS6.

In immunoblots, all four proteins were readily detected in total leaf extracts Fig. It should be noted that while this antiserum had high specificity for the kDa RPS6 in maize roots [ 66 ], numerous cross-reactive proteins were detected in tomato leaves. However, the kDa RPS6 was the most strongly detected protein and was only identified in leaf homogenates total leaf protein. These immunoblot data indicate that the chloroplasts are largely free of cytosolic protein contamination.

Our proteomics data also supports this result as the cytosolic RPS6 was detected in two of our eight samples with one unique peptide Additional file 3 : Table S3. Small amounts of the lumenal OEC23 were also detected in the non-soluble fraction, consistent with OEC23 being an extrinsic protein that associates with OEC33 and OEC16 at the thylakoid membrane for their role in oxygen evolution [ 90 , 91 ].

Both stromal- and lumen-localized proteins HSP70 and OEC23, respectively were detected in the stromal protein samples via immunoblots [ 90 — 92 ]. These data suggested that the stromal extract contained soluble lumenal proteins. The chloroplast lumenal proteome is not complex ranging from 80 to proteins [ 93 — 95 ]; 45 proteins designated as thylakoid peripheral or lumenal proteins in PPDB [ 77 ] were detected in the stromal proteome, representing 3.

In five samples, proteins were acetone precipitated. Manual curation of these proteins was performed using our tomato chloroplast protein Atlas [ 68 ], which predicted tomato protein localization using five published algorithms [ 72 — 76 ]. The proteins identified in the tomato chloroplast stromal extracts are shown based on their designated categories.

The chloroplast stromal proteome has chloroplast proteins [ 68 ]. There were co-isolating proteins CIPs that were reproducibly detected. In the eight stromal samples, proteins were detected once with 1 PSM. Of these proteins, 83 proteins were known to be located within the chloroplast.

The remaining proteins had no evidence for chloroplast localization and were classified as low-level contaminants and were removed from further consideration Fig. Using conservative criteria to identify stromal proteins, we removed proteins identified by one unique peptide Additional file 3 : Table S3; Fig.

These proteins had no empirical data to support their localization in the chloroplast based on Arabidopsis homologs PPDB, plprot and SUBA4 evidence or the tomato chloroplast protein Atlas. In addition, proteins were identified by more than one peptide but were detected sporadically one to three times in our eight samples ; these proteins were designated as low-level contaminants and not considered further Additional file 4 : Table S4; Fig.

In some cases, the PSMs for the sporadically identified proteins were high. A summary of the subcellular localization of the proteins identified by one peptide and the sporadically identified proteins is provided in Table 2. Their distribution in the cytosolic, endomembrane, nuclear, mitochondrial, peroxisomal, and plasma membrane compartments was similar.

Comparison of deduced protein localization for the co-isolating proteins, proteins detected by one unique peptide, and sporadically identified proteins. These CIPs could: 1 be reflective of the inadvertent co-isolation of small quantities of other organelles; 2 report the extensive and dynamic interactions of chloroplasts with other organelles e.

We assessed the frequency of detection, abundance, and putative localization of the CIPs Additional file 5 : Table S5. For example, of the CIPs For perspective, the range of SAFs for the tomato proteins was from 0.

CIPs predicted to be localized in the cytosol, peroxisome, nucleus, mitochondrion, and endomembrane system are shown. Each circle represents a single protein. The predicted subcellular localization of CIPs was imputed based on the tomato chloroplast Atlas, which provided predictions of the locations of the tomato CIPs Additional file 5 : Table S5.

Collectively these data indicated that a majority of the tomato CIP proteins were predicted to reside in the cytosol However, all three of these Arabidopsis proteins were also detected in other subcellular locations and the tomato Atlas did not predict a chloroplast localization, hence the classification as tomato CIPs.

This prediction was not supported by empirical data for the Arabidopsis CIP homologs, as 31 of these Arabidopsis homologs had a non-chloroplast location Additional file 5 : Table S5. Embedded within the cytosol, contamination of chloroplasts with abundant cytosolic proteins is anticipated.

In pea, PGKc co-localizes with glyceraldehydeP-dehydrogenase, triose-P-isomerase and aldolase providing an opportunity for direct channeling of substrates between the enzymes [ ]. However, these additional enzymes were not identified as stromal CIPs suggesting that this complex does not exist in tomato or the protein associations are labile. Of the remaining cytosolic CIPs, proteins associated with numerous functions were identified.

The Arabidopsis AKR2A homolog At2g works with a cytosolic HSP17 to target membrane proteins to the plastid outer membrane [ — ]; however, the tomato cytosolic HSP17 homolog was not detected in any of our stromal samples. The most highly represented cytosolic CIPs were those associated with translation, with four elongation factors, two initiation factors, two ribosomal protein subunits, and five tRNA synthetases Additional file 5 : Table S5.

When the lists of sporadically identified proteins and proteins identified with one unique peptide were examined, an additional 38 ribosome subunits, five initiation factors and three elongation factors were also identified Additional file 3 : Table S3, Additional file 4 : Table S4. Chloroplasts, peroxisomes, and mitochondria participate in the photorespiratory pathway that catabolizes the products produced by the oxygenation reaction of ribulose-1,5-bisphosphate carboxylase [ ].

Electron microscopy and in situ laser analyses have shown that in the light, peroxisomes and mitochondria have intimate and dynamic interactions with chloroplasts and with each other [ , ]. Chloroplasts may also interact with peroxisomes via dynamic peroxisome membrane extensions called peroxules [ ]. Therefore, it is not surprising that 19 peroxisomal proteins and 52 mitochondrial proteins were CIPs Additional file 5 : Table S5, Fig.

Hydroxypyruvate reductase Solyc01g , Glutamate:glyoxylate aminotransferase Solyc05g , and Serine:glyoxylate aminotransferase Solyc12g were identified in all eight samples and were abundant proteins with NSAF scores of 0. A byproduct of photorespiration is hydrogen peroxide, which is dissipated by a robust peroxisomal ROS-scavenging system [ ]. Reumann et al. Although we are extrapolating between two species, the substantial differences in NSAF values for peroxisomes determined by Reumann et al.

In tomato, ICL is detected in fruits and leaves [ ] and has been correlated with the peroxisome to glyoxysome transition during leaf senescence [ ]. It is noteworthy that other enzymes of the glycolytic cycle e. NSAFs ranged from 1. Proteins associated with the TCA cycle 14 proteins and amino acid biosynthesis or catabolism 14 proteins were enriched in the CIPs. There is substantial evidence that nuclei and plastids interact [ ]. Chloroplasts can be found directly appressed to nuclear envelopes and connected to nuclei via stromules.

These direct and yet dynamic communication channels may allow for the exchange of metabolites, H 2 O 2 , and, perhaps, proteins. Twelve chromatin-associated proteins i. Finally, there is a well-established biochemical continuity between the endoplasmic reticulum and the chloroplast [ 31 ]. Therefore, it is not surprising that there were 45 endomembrane system proteins that were identified as CIPs Table 2 , Additional file 5 : Table S5, Fig.

Tomato is the most cultivated horticultural crop worldwide, with over 4. In addition, tomato is a model system for the study of the induction of plant defenses associated with wounding, herbivory and pathogen attack [ ]. As chloroplasts are key regulators of stress perception and signal transduction [ 5 , 33 ] and the site of production of secondary metabolites and plant hormones involved in defense, an understanding of the dynamics of the chloroplast leaf proteome is needed.

The protocol provided here provides a detailed method to assure high quality and high yields of intact chloroplasts from tomato leaves suitable for proteomics analysis. As a number of yield-limiting steps were identified in this protocol, the methods can be adapted to virtually any plant species. In conjunction with the tomato nuclear and plastid genome sequences [ 56 , 71 ], evaluation of changes to the tomato chloroplast proteome, and its sub-organellar fractions, in response to cues during development, as well as abiotic and biotic stress are now possible.

Future confirmation of CIP localization using fluorescent reporter fusion proteins will determine if these proteins are imported and localized in more than one organelle or if their co-isolation with chloroplasts solely reflects the known tight apposition of ER, peroxisomes, mitochondria, and nuclei with chloroplasts [ , , , ].

Meenakshi Kagda UC Riverside. We thank Dr. Kenneth Cline U Florida and Dr. OB and LLW wrote the manuscript collaboratively. All authors read and approved the final version of the manuscript. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Oindrila Bhattacharya, Email: ude. Irma Ortiz, Email: ude.

Linda L. Walling, Email: ude. Supplementary information accompanies this paper at Plant Methods. Published online Sep Author information Article notes Copyright and License information Disclaimer. Corresponding author. Received Aug 7; Accepted Sep 4. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.

If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

This article has been cited by other articles in PMC. Protocol comparison. Additional file 2: Table S2. Chloroplast Isolation Interactive Worksheet. Additional file 3: Table S3. Proteins detected based on one unique peptide. Additional file 4: Table S4. Proteins that were sporadically identified. Additional file 5: Table S5. Co-isolating proteins CIPs. Abstract Background Chloroplasts are critical organelles that perceive and convey metabolic and stress signals to different cellular components, while remaining the seat of photosynthesis and a metabolic factory.

Results With the long-term goal of understanding chloroplast proteome dynamics in response to stress, we describe a high-yielding method to isolate intact tomato chloroplasts and stromal proteins for proteomic studies. Conclusions Our optimized method for chloroplast isolation increased the yields of tomato chloroplasts eightfold enabling the proteomics analysis of the chloroplast stromal proteome.

Background Plastids control key metabolic processes central to cell vitality and function. Materials and methods Note: This protocol is scaled for three g leaf chloroplast preparations. Proteinase inhibitor cocktail for plant cell and tissues extracts Sigma-Aldrich, catalog P Graduated glass cylinders one 1-L, one mL, two mL, one mL, sterile. Adapters for mL-centrifuge bottles four, Beckman-Coulter, catalog Nitrocellulose filters 0.

Glassware, plasticware, consumables, and equipment for isolation of stroma Beakers one mL. Polycarbonate ultracentrifuge tubes, 3. Wide-bore 1-mL pipette tips: Using a razor blade, cut 2 mm off the tip of 1-mL pipetman tips. A minimum of three wide-bore tips will be needed per chloroplast preparation. Wide-bore tips are stored in a 1-mL pipette-tip box and autoclaved. Stock solutions for chloroplast isolation 1 week in advance Note: For stock solutions for proteomics preparations, gloves are worn continuously to avoid common protein contaminants.

Adjust pH with NaOH. Table 1 Chloroplast and stromal protein yields. Open in a separate window. Leaf segments were added in two batches into the blender b The tomato leaf 2 chloroplast prep was performed with optimized blender time and speeds. All that changes when one of Michael's high school students accuses him of 'in All that changes when one of Michael's high school students accuses him of 'inappropriate conduct', and the town rushes to judgment.

Sign In. Play trailer Drama Romance. Director Steven Williford. Paul Marcarelli screenplay Steven Williford story. Top credits Director Steven Williford. See more at IMDbPro. Trailer The Green. Top cast Edit. Jason Butler Harner Michael as Michael. Cheyenne Jackson Daniel as Daniel. Illeana Douglas Trish as Trish. Karen Young Janette as Janette. Bill Sage Leo as Leo. Xander Johnson Jock No. Tom Bloom George as George. Marcia DeBonis Brenda as Brenda. Mark Blum Stuart as Stuart.

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