Anti rabbit igg horseradish peroxidase hrp conjugate

To detect the activated form of MpkA, 1×10⁷ conidia were inoculated into 100 ml liquid complete medium and cultured at 37°C for 22 h or 50°C for 46 h. Cell wall damage was induced by the addition of 100 μg ml⁻¹ Congo red for 2 h. Mycelia were harvested, ground in liquid nitrogen to a fine powder using a mortar and pestle, and immediately suspended in lysis buffer (200 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1 mM phenyl- methanesulphonyl fluoride) and incubated on ice for 20 min. Proteins in the supernatant were collected by centrifugation (16 000 g at 4°C for 10 min). Protein concentration was determined by the Lowry method. Thirty micrograms of cellular proteins was separated by 12% SDS-PAGE and transformed to PVDF membrane (Millipore, USA). After blocking in 5% skimmed milk in Tris-buffered saline (TBS; 10 mM Tris-HCl, 150 mM NaCl, pH 8.0), the membrane was probed with anti-phospho-p44/42 MAPK antibody (Cell Signaling Technology, USA) in TBS containing 1% skimmed milk. The membrane was then washed for 15 min each in TBS plus 0.05% Triton X-100 (TBST), TBST plus 0.5 M NaCl and TBST. Anti-Mn-SOD antibody was used as control. The primary antibody was detected using Anti-Rabbit IgG Horseradish peroxidase (HRP) Conjugate (Promega) and Enlight™ reagents (Engreen Bio-system, China).