Electro transfer unit

Leaves from agroinfiltrated plants with recombinant A. tumefaciens and leaves from non-agroinfiltrated plants were ground in liquid nitrogen, suspended in extraction buffer [250mm sucrose, 50mm N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) buffer pH 8.0, 1 mm ethylenediaminetetraacetic acid (EDTA), 20mm β-mercaptoethanol, 0.06% L-cysteine, 1mm MgCl₂, 0.6% polyvinylpyrrolidone (PVP), 2% triton, and 1% phenylmethylsulfonyl fluoride (PMSF)], and incubated at 4°C overnight. After centrifugation (10,000g), the supernatants containing the total soluble proteins (TSP) were loaded in a nitrilotriacetic-acid-Ni²⁺ column (Ni-NTA; Qiagen, Germantown, MD, United States). The proteins that were retained through the column were washed with wash buffer (PBS pH 8.0, 10mm imidazole, 1% PMSF), and AtHsp81.2-SAG1_HC was eluted with elution buffer (PBS pH 8.0, 250mm imidazole, 1% PMSF). AtHsp81.2–SAG1_HC was purified by SDS-polyacrylamide gel electrophoresis (10% SDS-PAGE) using the Mini-Protean system III (Bio-Rad) and was either stained with Coomassie brilliant blue or transferred onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Wauwatosa, WI, United States) using an electro-transfer unit (Bio-Rad).

For Western blot analysis, purified protein was incubated at 100°C for 5 min in loading buffer, separated by SDS-PAGE (12% gel) and transferred onto PVDF membrane (GE) using an Electro-transfer unit (Bio-Rad). The membranes were sequentially incubated with mouse polyclonal anti-rTgPI-1 antibody (1:1000) or anti-Histidine (1:3000; Sigma) and alkaline phosphatase conjugated goat anti-mouse IgG (1:5000; Sigma). After washing, the reaction was developed by the addition of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrate. PageRuler^TM Prestained Protein Ladder (Thermo Scientific) was used as molecular marker.

Purified recombinant proteins were separated by SDS-PAGE (10%) using the Mini-Protean system III (Bio-Rad, Hercules, CA, USA) and stained with Coomassie Brilliant Blue. For Western blot analysis, recombinant proteins were transferred onto PVDF membranes (GE Healthcare, Chicago, IL, USA) using an Electro Transfer Unit (Bio-Rad). The membranes were incubated with either mouse anti-6xHIS monoclonal antibody (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA), mouse polyclonal antibodies against rSAG1m (1:1000) or sera from NbHsp90.3-SAG1_HC immunized mice (1:100) as primary antibodies. Then, membranes were incubated with alkaline phosphatase conjugated goat anti-mouse IgG (1:5000, Sigma, St. Louis, MO, USA). The reaction was developed by the addition of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Promega) substrate. PageRuler^™ Prestained Protein Ladder (Fermentas, Waltham, MA, USA) was used as molecular marker.