Es et al., 2010) and quantified employing sinigrin as the standard at 227 nm. Myrosinase activity Myrosinase activity was assayed as described by Barth and Jander (2006). Briefly, 30 mg of frozen leaves were ground with 5 extraction buffer (wv) [33 mM sodium phosphate, pH 7, 5 polyvinylpolypyrrolidone (PVPP), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM -aminocaproic acid, ten M leupeptin]. Next, 50 of extract (diluted 1:25) was incubated with 200 of reaction buffer (33 mM sodium phosphate, pH 7, 0.35 mM sinigrin, 0.33 mM ascorbic acid). Spectrophotometry was utilized to monitor sinigrin disappearance at 227 nm (25 , 15 min). Immunoelectrophoresis For -thioglucoside PbTx-3 Data Sheet glucohydrolase 1 (TGG1; myrosinase 1) and -thioglucoside glucohydrolase two (TGG2; myrosinase 2) content quantification, proteins had been extracted from 20 mg of leaf powder with 0.4 mL of extraction buffer (10 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 mM DTT, 0.1 Triton X-100, ten glycerol, 0.05 BSA, 0.5 PVPP, 50 mM HEPES, pH 7.five) within the presence of a cocktail of proteases inhibitors (1 mM PMSF, 1 mM -aminocaproic acid, 10 M leupeptin). Samples were then centrifuged at 4000g for 30 min at 4 and also the supernatants recovered. The protein content in the supernatants was quantified by a dye-binding protein assay (Bio-Rad Bradford Protein assay) with BSA because the regular for the calibration curve. Equal amounts of proteins were loaded onto a 1.five mm-thick denaturizing four.six (wv) stacking and ten (wv) resolving gel. Gels have been electroblotted onto a nitrocellulose membrane and blots blocked in 5 (wv) skim milk in 20 mM Tris-buffer saline at four for 1 h, washed, and incubated with -TGG1 or TGG2 inside a AG-494 References dilution of 1:5000 (Ueda et al., 2006). They have been then incubated with goat antirabbit horseradish peroxidase conjugate secondary antibody (1:20 000). Finally, immunoreactive bands had been visualized with a Molecular Imager ChemiDoc XRS System (BioRad) and quantified with ImageJ software. Sample preparation and labelling for proteomic analysis Fifty milligrams of leaves had been ground in liquid nitrogen and homogenized in 0.five mL extraction buffer [7 M urea, two M thiourea, four CHAPS, two Triton X-100, 50 mM DTT, and 0.five plant protease inhibitor and phosphatase inhibitors cocktails (SigmaAldrich)]. Samples have been centrifuged for 15 min (10 000g, 4 ) and total protein precipitated from 200 of supernatant with methanol and chloroform (600 methanol, 15 chloroform, and 450 ultrapure water). Mixtures have been vortexed and centrifuged for 1 min at 14 000g. The aqueous phase was then removed, an added 450 of methanol added, and centrifugation repeated. The methanol phase was removed plus the protein pellets dried inside a vacuum centrifuge and finally resuspended inside a option containing 7 M urea, two M thiourea, and four CHAPS (15 ). Protein quantification was performed with a dye-binding Bradford micro-assay (Bio-Rad), along with a shotgun comparative proteome-wide evaluation of total leaf extracts (4 biological replicates) was carried out making use of isobaric tags for relative and absolute quantitation (iTRAQ; Unwin et al., 2010). iTRAQ labelling was performed in accordance with the manufacturer’s protocol (Sciex). Briefly, 100 g of total protein was decreased with 50 mM Tris(2-carboxyethyl)phosphine at 60 for 1 h, and cysteine residues were alkylated with 200 mM methylmethanethiosulfonate (MMTS) at room temperature for 15 min. Protein enzymatic cleavage was carried out with trypsin (Promega; 1:20, w:w) at 37 for 16 h. An iTRAQ.
