Anti gfp primary antibody

The N. benthamiana leaf samples were harvested 48 h after infiltration. Total protein was extracted from powdered plant tissue using a phenol solution (0.5 M Tris pH 9.4, 50 mM EDTA, 0.7 M sucrose, 0.1 M KCl containing 2% ß-mercaptoethanol and complete protease inhibitors (Roche)). The protein concentration was measured using a Bradford assay following the manufacturer’s instructions (Bio-Rad). 0.1 mg protein of each sample was separated on Tris-Glycine SDS-PAGE gels and transferred to nitrocellulose membranes using an iBlot (semi-) dry blotting system from Life Technologies. The blotting membrane was blocked with 5% BSA (bovine serum albumin) in TBST solution (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. The membrane was incubated with 1:3000 diluted anti-GFP primary antibody (Sigma) for 3 h at room temperature. The membrane was washed with TBST solution and incubated with a 1:3000 dilution of anti-mouse IgG antibody (Sigma). The signal was detected using an AP conjugate substrate kit (Bio-Rad).

Cultures were grown to exponential phase in 4% glucose containing SD medium, transferred to 2% galactose containing medium and grown overnight. Then, three OD units were harvested by centrifugation and protein extracts were prepared by using an alkaline lysis method. The cells were permeabilized with 0.185 M NaOH plus 2% β-mercaptoethanol. After 10 min incubation on ice, trichloroacetic acid (TCA) was added to a final concentration of 5%, followed by an additional 10 min incubation step on ice. Precipitates were collected by centrifugation for 5 min at 13000 g and pellets were resuspended in 50 μL of sample buffer (4% sodium dodecyl sulfate, 0.1 M Tris-HCl pH 6.8, 4 mM EDTA, 20% glycerol, 2% β-mercaptoethanol, and 0.02% bromophenol blue) plus 25 μL of 1 M Tris-Base. Samples were separated by standard SDS-PAGE on 12% polyacrylamide gels and further analyzed using standard Western blotting techniques. An anti-GFP primary antibody (Sigma-Aldrich) and anti-Mouse (GAM)-HRP conjugated secondary antibody (Biorad) were used. The ECL method (SuperSignal West Pico or Femto, Thermo Scientific) was used for detection and visualization of the blots was performed with a UVP Biospectrum^® Multispectral Imaging System.

Tobacco leaves (~ 10 mg) sampled 3 days after infiltration were ground in 150 µl sample buffer (Wako Pure Chemical Industries, Osaka, Japan) containing 5% β-mercaptoethanol. Samples were denatured at 98 °C for 2 min and proteins were separated by 10% polyacrylamide gel (SuperSep™Ace, Wako Pure Chemical Industries) electrophoresis. Proteins were transferred to PVDF membranes (Immobilon, Millipore) by semidry electroblotting. Blots were probed with anti-GFP primary antibody (SAB4301138, Sigma-Aldrich Co. LLC) and alkaline phosphatase conjugated anti-mouse secondary antibody (A3562, Sigma-Aldrich Co. LLC) and visualized using WesternBlue^® Stabilized substrate for alkaline phosphatase (Promega).