Plant total protein extraction kit

Total soluble protein was extracted from the seeds at 30–40 days after pegging (DAP) of control FH1 and the transgenic T₃ generation lines using a plant total protein extraction kit (Sangon Biotech Co., Ltd., Shanghai, China). Total protein extracts of over 40–50 μg for each sample were boiled for 5 min in 6× loading buffer (Transgen Biotech, Beijing, China), separated by 10% sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a 0.45-μm polyvinylidene difluoride (Roche Applied Science, Mannheim, Germany) membrane by the wet electrotransfer method (Tovey and Baldo, 1987 (link)). After transfer, blotting efficiency was checked by reversibly staining the transferred proteins with Ponceau S solution. Using mouse monoclonal antibody Myc-Tag (9B11; Cell Signaling Technology, Inc.) as the primary antibody, western blotting was carried out in accordance with the instructions of the Lumi-Light^PLUS Western Blotting Kit (Roche Applied Science).

The shoots (10 cm) from plants of which scions from J. gossypifolia, J. integerrima, J. multifida or J. podagrica were grafted onto WT and SUC2:JcFT seedlings grown in an artificial climate chamber were collected to isolate their total protein. Apical and lateral buds from different scion positions were collected and flash frozen in liquid nitrogen. After grinding, the powder was dissolved in 1 ml of extraction buffer to extract the proteins. The total soluble proteins were isolated using a Plant Total Protein Extraction Kit (No. C500053, Sangon Biotech, Shanghai, China) following the manufacturer’s instructions. The protein concentration was subsequently measured using a Lowry Protein Assay Kit (No. C504031, Sangon Biotech) according to the manufacturer’s instructions.

Total proteins were extracted from the stolons of the bermudagrass plants using the Plant Total Protein Extraction Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The protein concentration was also quantified using the Bradford method. For trypsin digestion, 100 μg of protein was firstly reduced with 10 mM DTT for 45 min at 50 °C and alkylated with 50 mM iodoacetamide for 45 min at room temperature in the dark. The protein solution was then diluted with four volumes of digestion buffer (100 mM TEAB, pH 8.0) containing 2 μg of Trypsin Gold (Promega, Madison, USA) and incubated at 37 °C overnight. The digested peptides were desalted using a Strata-X C18 SPE column (Phenomenex, Torrance, USA) and lyophilized by vacuum centrifugation. Acetylated and succinylated peptides were enriched from the lyophilized peptides using the PTMScan® Acetyl-Lysine Motif [Ac-K] Kit (Cell Signaling Technology, Danvers, USA) and PTMScan® Succinyl-Lysine Motif [Succ-K] Kit (Cell Signaling Technology) according to the manufacturer’s instructions, respectively.