Horseradish peroxidase hrp conjugated goat anti mouse igg secondary antibody

Genomic DNA (gDNA) of P. capsici was extracted using a TIANGEN DNAsecure Plant kit (Beijing, China) and RNA was removed by RNase treatment and column chromatogram. Equal amounts of gDNA were denatured at 95°C for 5 min and chilled on ice for 10 min. DNA were spotted onto Amersham Hybond-N⁺ membranes (GE Healthcare, Beijing, China), and further dried at 37°C for 30 min. DNA was then crosslinked under UV for 5 min. The membrane was blocked in 5% milk phosphate-buffered saline with Tween 20 (PBST) for 1 h and incubated with 6mA antibody (sysy, 202003) or 5mC antibody (Abcam, ab73938) in 5% milk PBST overnight at 4°C. After a PBST wash, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (Proteintech, Beijing, China) for 1 h and treated with ECL substrate (CWBIO, Jiangsu, China). After washing, the signal was detected by Tanon 5200 (Shanghai, China). For input quantification, the same membrane was incubated with 0.1% methylene blue solution for 15 min and washed using tris-buffered saline with Tween 20 (TBST) buffer three times. Relative 6mA abundance was quantified (integrated signal density anti-6mA/integrated signal density input DNA) using ImageJ (NIH image to ImageJ: 25 years of image analysis.).

The purified fusion protein was solubilized in sample buffer (50 mM Tris·HCl pH 6.8, 10% glycerol, 0.1% bromophenol blue, 1% 2-mercaptoethanol, 2% SDS) and loaded onto SDS polyacrylamide gels. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membrane (Roche Diagnostics) for 2 h at 100 V using a transblotting apparatus (Bio-Rad, USA). The membrane was blocked overnight at 4 °C in 5% skimmed milk-TBST and was detected by GST-Tag monoclonal antibody (3A10) (Bioworld Technology, China) with a 1:8000 dilution at RT for 1 h. The primary antibody binding was incubated with a 1:20000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (Proteintech, China) at RT for 1 h and visualized with an enhanced chemiluminescence kit (CWBio, China).