Quickblock secondary antibody dilution buffer for western blot

The binding capacity of each Fab to knob proteins was determined by western blot. Ten micrograms of purified knob proteins were preheated (100 °C for 10 min) and then separated and analyzed by electrophoresis on 10% SDS-PAGE under denaturing and reducing conditions, and transferred onto polyvinylidene fluoride membranes. The membranes were then blocked with QuickBlock Blocking Buffer for Western Blot (Beyotime Biotechnology, Shanghai, China) overnight. After washing again with PBS, Fab (5 μg/mL) in QuickBlock Primary Antibody Dilution Buffer for Western Blot (Beyotime Biotechnology, Shanghai, China) was added and incubated at 25 °C for 2 h. After washing thrice with 0.05% PBST, bound Fab was detected by incubation with a 1:5000 dilution of HRP-conjugated anti-HA tag mouse mAb (Abbkin, Redlands, CA, USA) in QuickBlock Secondary Antibody Dilution Buffer for Western Blot (Beyotime Biotechnology, Shanghai, China) at 25 °C for 1 h, and washed thrice in 0.05% PBST. The blot was developed by the ECL Detection System (UVP Chemstudio, Analytik Jena AG, Thuringia, Germany).

The purified rGST-On-CRP was loaded on 12% SDS-PAGE and transferred to a PVDF membrane (IPVH00010, Merck, Darmstadt, Germany) and then blocked with QuickBlock™ Blocking Buffer for Western Blot (Beyotime, Shanghai, China) at 25 °C for 15 min. The membrane was then incubated with anti-GST-tag antibodies (AG768, Beyotime, Shanghai, China), diluted at a ratio of 1:1000 in QuickBlock™ Primary Antibody Dilution Buffer for Western Blot (Beyotime, Shanghai, China) and incubated at 25 °C for 1 h.
Then the membrane was washed three times in TBST (TBS + 0.1% Tween 20) and incubated with secondary antibody HRP-labeled goat anti-rabbit IgG (H + L) (A0208, Beyotime, Shanghai, China), diluted at a ratio of 1:2000 in QuickBlock™ Secondary Antibody Dilution Buffer for Western Blot (Beyotime, Shanghai, China) at 25 °C for 30 min. Finally, the membrane was washed three times in TBST, and the antigen–antibody complexes were detected via the enhanced chemiluminescence method (P0018S, Beyotime, Shanghai, China).