Anti gfp antibody

Total protein was extracted from transgenic plants containing the construct Pro_AAP1-AAP1CDS-GFP using cell lysis buffer for western blot and immunoprecipitation (Beyotime P0013). Immunoblot analysis was performed using an anti-GFP antibody (Abmart) and Anti-Mouse IgG (Abmart).

The tissue was ground in liquid nitrogen, and total protein was extracted using 2×SDS loading buffer. The samples were resolved on a 12% SDS-PAGE gel and transferred onto an Amersham Hybond-P PVDF membrane (GE Healthcare) using Tris-Gly transfer buffer. Membranes were blocked with 5% (w/v) milk in 0.05% (v/v) TBS-plus Tween 20 (TBST) for 40 min and incubated overnight at 4°C with 1:5,000 dilutions of primary antibodies of mouse anti-Flag conjugated to horseradish peroxidase (Abmart), washed three times with TBST, and incubated overnight at 4°C with 1:1,000 dilutions of secondary antibodies (Beyotime). The membranes were then washed three times with TBST. Detection was performed using ECL Plus western blotting Detection Reagents (GE Healthcare) and ChemiDoc Touch Imager (Bio-Rad). Anti-GFP antibody (Abmart) was used to determine the expression level of the YFP protein. Ponceau was used as a loading control, and MAPK assays were performed as previously described (Zhang et al. 2021 (link)). Briefly, 1 mm of flg22 peptide was infiltrated into the leaves of 4-week-old plants. Total protein samples were collected at 0, 5, 10, 20, and 30 min and used for immunoblotting with an anti-p44/42 mAPK antibody (Cell Signaling Technology) to detect activated forms of MPKs. Image data were analyzed using Image Lab Software (Bio-Rad) and assembled using Adobe Photoshop CS6.

As previously described, we transfected apple calli using recombinant plasmids pRI-MdERF1B and pRI-MdEIL1. Then, we conducted ChIP-qPCR assays with an EZ-ChIP Kit (Millipore/Upstate, Temecula, CA, USA) and an anti-GFP antibody (Abmart, Shanghai, China) according to a previous description by Wang et al. [46 (link)]. Then, we used qPCR data to determine the amount of immunoprecipitated chromatin. All ChIP assays were performed in triplicate.

Apple calli or plantlets were ground into powder in liquid nitrogen and immediately immersed in protein extraction buffer [100 mM NaCl, 0.5 mM EDTA, 20 mM Tris-HCl (pH 7.5), 0.5 mM PMSF, 0.5% Nonidet P-40 and 0.5% protease inhibitor] for 15 min on ice. The mixture was then centrifuged at 12,000 rpm for 15 min at 4 °C. The total protein supernatant was collected and separated on a 10% SDS-PAGE gel, transferred to a polyvinylidene fluoride (PVDF) membrane (Roche, IN, USA), and probed with anti-GFP antibody (Abmart, Shanghai, China). Western blotting assays were conducted as previously described^{30 (link)}. ACTIN served as a protein loading control.

Total proteins were extracted from the leaf infiltrated area as previously described (47 (link)). Protein samples were fractioned by 10% SDS-PAGE gel and electroblotted onto polyvinylidene difluoride (PVDF) membrane, then immunoblotted with anti-GFP antibody (Abmart, China) at a dilution of 1:5,000. HiSec horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) (Nanjing Vazyme Biotech Co., Ltd., China) was used as the secondary antibody. The chemiluminescence signals were detected with a FDbio-Femto RCL kit (FDbio Science, China).