Hrp conjugated goat anti mouse secondary antibody

Protein was extracted from the mycelia by radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, USA) for western blotting. The supernatants were collected after centrifugation at 10,000 × g for 5 mins and mixed with 5 × loading buffer (60 mmol/L Tris–HCl, 2% SDS, 25% glycerol, 5% 2-mercaptoethanol, and 0.2% bromophenol blue) at a ratio of 1:4. Samples were then heated at 95°C for 5 min and separated on 5% SDS-polyacrylamide gel. The separated proteins were then transferred to a 0.22 μm polyvinylidene difluoride (PVDF) membrane (Perkin Elmer, MA, USA). The primary antibody used for western blotting was Guide-it™ Cas9 Monoclonal Antibody (Takara, Beijing, China). The secondary antibody was HRP-conjugated Goat Anti-Mouse Secondary Antibody (ABmart, Shanghai, China).

The agro-infiltrated leaf tissues were homogenized in 500 μl of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 2 mM Na₃VO₄, 25 mM β-glycerophosphate, 10 mM NaF, 0.05–0.1% Tween, 1 mM PMSF, pH 7.5). The homogenates were centrifuged at 14,000 × g for 20 min at 4 °C, then the supernatants were collected and the protein concentration was determined by the Bradford method^{64 (link)}. Aliquots of 50 μg of protein were separated on 8% acrylamide gels and were then electro-transferred to PVDF membranes using the Bio-Rad gel transfer apparatus in TBST buffer for 1 h. Membranes were blocked in a 3% BSA/TBST buffer solution at room temperature for 0.5 h, followed by incubation with the appropriate primary antibodies at 4 °C overnight [anti-FLAG, Abmart, cat # M20008 at 1:1000 dilution; anti-HA, Roche, cat # 12013819001 at 1:1000 dilution; anti-c-Myc, Millipore, cat # 05-724 at 1:1000 dilution). Following incubation, the membranes were washed three times with TBST and incubated with an HRP-conjugated goat, anti-mouse secondary antibody (Abmart, lot # M21001 at 1:1000 dilution). The blots were washed again three times with TBST buffer and the immunoreactive bands were detected using an enhanced chemiluminescence method.