Anti egfp antibody

The CDS of TaMYB4 and TaMDHAR were constructed into the pCAMBIA2300-EGFP vector. Tobacco leaves (30 days old) were then injected with Agrobacterium tumefaciens GV3101 harboring pCAMBIA2300-EGFP, pCAMBIA2300-TaMYB4-EGFP or pCAMBIA2300-TaMDHAR-EGFP plasmids. After 72 h of injection, the leaves were harvested to observe the EGFP signal under a fluorescence microscope. Western blot analysis was performed to further analyze the subcellular localization results. The detailed experimental methods refer to those used by the authors in [43 (link)]. Anti-EGFP antibody (1:1000, Proteintech, Wuhan, China) was used as the primary antibody to probe EGFP. Horseradish peroxidase (HRP)-conjugated anti-rabbit antibody was chosen as the secondary antibody (1:2000, Proteintech).

The expression of EGFP was evaluated by Western blot. The 10 μL of supernatant was loaded into each well of 10% SDS-PAGE gel. After finishing the electrophoresis, the proteins in the gel were transferred to Hybond-C nitrocellulose membrane (Amersham Bioscience). The transfer was done at 100 V for 2 h. Anti-EGFP antibody (Proteintech, China, Cat no. 50430-2-AP) and IRDye 800CW-conjugated goat anti-rabbit secondary antibodies (LI–COR Biosciences, Lincoln, NE, USA; cat. no. C60607-15) were employed as the primary and secondary antibody, respectively. The hybridization signals were detected and measured using LICOR Odyssey system (LI–COR, Nebraska, USA).

The expression level of eGFP in L102 and L322 was determined by Western Blot as described previously [42 (link)]. Briefly, spores were inoculated into 35 mL of seed medium for 20 h in a 250 mL flask. Then cell density (OD_600nm) was determined by UV spectrophotometry. Seed culture was transferred into 35 mL of TSB medium in a 250 mL flask, set up with a starting OD_600nm of 0.15. After incubation at 30 °C for 12 h, 24 h, 36 h, 48 h in a rotary shaker, 500 μL mycelia were collected and washed once with 1 mL PBS buffer, finally re-suspended in 500 μL PBS buffer. The mycelia suspensions were sonicated on ice (4 × 5 s, with 5 s intervals every time). The samples were centrifuged at 12,000 rpm for 10 min at 4 °C and the supernatants were transferred to a 1.5 mL sterile EP tube. The total protein quantification was performed by Bradford assay. Then 15 μg of total protein were separated in 12% SDS-PAGE and western blot analysis was performed with rabbit polyclonal anti-EGFP antibody (Proteintech, USA).