Peirce ecl western blotting substrate

Samples were run on precast Tris-Glycine 8% Novex gels (Thermo Fisher Scientific XP00080BOX) with GTS running buffer (Tris 25 mM, Glycine 190 mM, SDS 0.1%) or 4–12% Bis-Tris precast NuPAGE gels (Thermo Fisher Scientific NP0322BOX) with MOPS running buffer (Thermo Scientific NP0001). For whole cell lysates, 60μg of protein was loaded. For cellular fractions, 60μg or 30μg of protein was loaded for the soluble or chromatin fractions, respectively. Proteins were transferred to PVDF membrane using the Iblot 2 system (Thermo Fisher Scientific IB21001). Membranes were then blocked for 1–2 hours in 5% powdered milk in PBST. Membranes were incubated overnight at 4°C with diluted antibodies as indicated. Membranes were then washed in PBST for 5 minutes 4X, followed by incubation with appropriate HRP conjugated secondary antibody at 1:10000–1:50000 dilution for 1 hour at room temperature. Membranes were again washed in PBST 4X 5 minutes each, followed by chemiluminescence either with Peirce ECL western Blotting Substrate (Thermo Fisher Scientific 32106), SuperSignal West Pico Plus (Thermo Fisher 34580), or SuperSignal West Dura extended Duration Substrate (Fisher Scientific 37071)

Total proteins were isolated from the hippocampal tissues 3 d post-TBI using RIPA protein lysate (Beyotime, Shanghai, China) and quantified using the bicinchoninic acid (BCA) assay kit (Beyotime, Shanghai, China). Five rats in each group were employed for Western blot analysis. The protein was separated by 10% polyacrylamide-SDS gels and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat dry milk at 4°C overnight and then incubated with primary antibodies (1 : 1000, Santa Cruz, CA, USA) at 4°C overnight. Next, the membranes were incubated with a horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 5000, ABclonal, Wuhan, China) at room temperature for 2 h. Finally, the blots were developed using Peirce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA). The protein expressions were normalized to β-actin expression.