One step plant active protein extraction kit

Sorghum (Sorghum bicolor (L.) Moench) seeds were germinated at 24 + 1 °C under a 16 hour (h) light/8 h dark cycle and seedlings were then transferred to pots in glasshouse conditions. One week after anthesis, the tissues used in the analysis were collected to extract protein for PME activity analysis.
Total protein extracts were obtained using a One-Step Plant Active Protein Extraction Kit (Sangon Biotech, C510004, Taiwan, China) and calibrated with Easy Protein Quantitative Kit (Bradford; TransGen, DQ101, Beijing, China) using bovine serum albumin (BSA) standard solution and Commas Brilliant Blue solution. The same amount of protein from different tissues were used to analyze. The detailed method was followed as previously reported [12 (link)]. Protein extracts (10 μg) in a same volume (20 μL) were loaded in to the 4 mm-diameter wells in 1% agarose gels containing citrus fruit pectin (0.1% (w/v; ≥85% esterified, Sigma-Aldrich, P9561, Germany), citric acid (12.5 mM) and 50 mM Na₂HPO₄, having pH 6.5. The gels were kept overnight at 28 °C followed by staining for 1 h with 0.05% (w/v) ruthenium red and washed for 4 h in water. The stained gels were photographed and the intensity of staining quantified with ImageJ software [14 (link)]. Measurements were performed in triplicate and data were normalized with the tissue stem area set to one.

N. benthamiana leaves inoculated with A. tumefaciens for 72–96 h were ground using liquid nitrogen. Protein extraction was performed using a one-step plant active protein extraction kit (Sangon Biotech, Shanghai, China) according to the instructions. The extracted proteins were separated using electrophoresis with 12% sodium dodecyl sulfate-polyacrylamide. To observe sample loading, the separated proteins were electrophoretically blotted on nitrocellulose membranes and stained using 0.1% Ponceau S. Five percent skimmed milk in TBS-T buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) was used to block the membranes for 1 h at room temperature, followed by incubation for 1 h using anti-FLAG antibody solution (1:5, 000 dilution) at room temperature; the membranes were then washed using TBS-T buffer. Blots were incubated for 1 h with horseradish peroxidase-conjugated anti-mouse secondary antibody (1:5, 000 dilution in TBS-T) at room temperature. The immunoblots were incubated using the eECL western substrate and observed with X-films.

Polypeptides of TAT-HezK I (YGRKKRRQRRRGGGGSYFSPWGa) and middle amino acids of CpTI and (GSNHHDDSSDEPSESSEPCCDSCa) were synthesized (Genscript, Nanjing, China), and polyclonal antibodies (anti-TAT-HezK I and anti-CpTI) were generated in rabbits, as described previously [21 ]. Western blot analysis of the transgenic plant materials was performed. Total soluble protein (TSP) was extracted from leaves harvested from six week old plants, 3–5 nodes below the apex. Tissues were ground to a fine powder in liquid nitrogen with a chilled mortar and pestle. TSP was extracted using the One Step Plant Active Protein Extraction Kit (Sangon) according to the manufacturer’s instructions. Concentration of TSP was determined by the BCA Protein Assay Kit (Sangon) according to the manufacturer’s protocol. For the Western blot, 20 micrograms of TSP was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 1× SDS running buffer (Sangon) at 80 V for 2 h. Proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The primary antibodies (anti-TAT-HezK I and anti-CpTI) were used at 1:5000 dilution, and the IgG-AP was used at a 1:20,000 dilution. Detection was performed using the NPT/BCIP kit (CWBIO company, Beijing, China) as described by the manufacturer.