Trkb antibody

A protein loading buffer of 5×SDS-PAGE was added to the sample at a ratio of 4:1. After denaturing at 100 °C for 5 min, the sample was stored in a −20 °C freezer until use. At the time of use, protein extracts (50 μg) were electrophoresed in 30% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Blots were blocked in PBST buffer (2 L ddH₂O and 2 mL of 0.05%Tween) with 5% dry milk and incubated with an anti-BDNF antibody (Abcam Biotechnology; 1:2,000), TrkB antibody (Cell Signaling Technology; 1:1,000), CREB antibody (CST; 1:1,000), and β-tublin antibody (Multisciences (Lianke) Biotech Co., Ltd; 1:1,000), and then incubated overnight at 4 °C. Blots were then rinsed 3 times with 1×PBST, for 10 min/time. The secondary antibody, Pierce Goat Anti-Rabbit IgG (Santa Cruz Biotechnology; 1:2,000), was added, and the blot incubated for 1 h at room temperature on a decolorizing shaker. It was then rinsed 3 times with 1×PBST, for 10 min/time. The colour developing solution A and B was mixed, and 1 mL added to the membrane. A ChemiScope 3,000 chemiluminescence instrument was used to detect and take pictures. The integral optical density value of the target protein was calculated and then compared with β-tubulin expression to give the degree of expression of the target protein.