Transient expression of two selected viral proteins fused to GFP, N, and NSs following agroinfiltration was validated by western blot. Soluble proteins from agroinfiltrated leaf tissues at 2 dpi (for localization assay) or 3 dpi (suppression of RNA silencing assay) were extracted in protein sample buffer (Laemmli, 1970 (link)) and separated on a SDS-12% polyacrylamide gel. Proteins were transferred to PVDF membrane (Millipore) and detected using mouse anti-GFP antibodies (1:1000, Roche Life Science) or mouse monoclonal anti-Flag M2 clone 2 antibody (1:2000, Sigma–Aldrich) followed by rabbit anti-mouse IgG horseradish peroxidase conjugate (1:25,000), Pierce ECL Plus chemiluminescent substrate, and exposure to x-ray film (Fujifilm). Protein sizes were estimated using pre-stained Page Ruler protein ladder (Thermo Fisher Scientific).
Pierce ecl plus chemiluminescent substrate
Manufactured by Fujifilm
Pierce ECL Plus chemiluminescent substrate is a laboratory reagent used for the detection of proteins in Western blotting applications. It is a solution that generates a luminescent signal when combined with a peroxidase-labeled detection system, allowing for the visualization of target proteins.
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2 protocols using pierce ecl plus chemiluminescent substrate
1
Viral Protein Expression and Detection
2
Agro-infiltration and Western Blotting
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Agro-infiltrated N. benthamiana leaves were harvested at 2 to 6 d post infiltration (dpi), and total proteins were extracted in SDS buffer [100 mM Tris (pH 6.8), 20% glycerol, 4% SDS, and 0.2% bromophenol blue, 10% β-mercaptoethanol] for western blotting analyses. Proteins were separated by SDS-PAGE gels followed by either staining with Coomassie blue for equal loading control or transfer to nitrocellulose membranes to detect accumulation using polyclonal antiserum specific to the BYSMV N (1:3000), P (1:3000), GFP (1:2000), RFP (1:2000), or HA (1:3000) proteins, followed by treatment with goat anti-rabbit/mouse IgG horseradish peroxidase conjugate (Bio-Rad, 1:3000), Pierce ECL Plus chemiluminescent substrate, and exposure to X-ray films (Fujifilm).
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