Ap conjugated affinipure goat anti rabbit igg

Total proteins from 10-d-old WT and val1 seedlings were isolated using a Plant Total Protein Extraction Kit (Sangon Biotech). An equal amount of protein was separated on 10% SDS-polyacrylamide gels. The proteins were transferred to iBlot 2 PVDF Regular Stacks using the iBlot 2 Gel Transfer Device (Invitrogen), immunoblotted with various primary antibodies (Agrisera and Beijing Protein Innovation Company) and AP-conjugated Affinipure Goat Anti-Rabbit IgG (Jackson ImmunoResearch) according to the recommended dilution ratios in the iBind Cards using the iBind Western Device (Invitrogen), and detected using the NBT/BCIP substrate (Sangon Biotech).

Plant leaf tissues were ground in protein sample buffer at a 1/50 dilution, denatured by boiling for 3 min, put on ice for 1 min and centrifuged at 16,110 g for 3 min. The supernatants were collected and separated in 12% SDS-PAGE, and then transferred onto nitrocellulose (NC) membranes in transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) at 120 V for 30 min. The NC membranes were washed with TSW buffer (10 mM Tris-HCl, pH 7.4, 154 mM NaCl, 0.25% gelatin, 0.1% Triton X-100 and 0.02% SDS) for 3 times, each time for 3 min, and then shaken with TSW buffer-diluted antibodies at room temperature for 30 min. After washing for 3 times with TSW buffer, each for 3 min, the NC membranes were incubated at room temperature with AP-conjugated affinipure goat anti-rabbit IgG (Jackson Immuno Research Laboratories) at a 1/5000 dilution in TSW buffer for 30 min. The NC membranes were then washed twice with TSW buffer for 3 min and rinsed with substrate buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl and 5 mM MgCl₂) for 3 min. Color development was conducted by adding 50 µl of 50 mg/ml NBT (nitro blue tetrazolium chloride) and 25 µl of 50 mg/ml BCIP (5-bromo-4-chloro-3-indoyl phosphate) in 7.5 ml substrate buffer. The reaction was stopped by submerging the NC membranes in water.