Optimization of blocking buffer for Western blot analysis of TrkB pY817 expression in NSC34 RIPA cell lysate (10 μg/lane) and mouse cortex homogenate (30 μg/lane), using rabbit antibody R-1717-50. NSC34 cells were grown in proliferation medium in presence and absence of Retinoic Acid (RA, 1 μM) for 5 days. Cells were then serum-starved for 4 hours and treated with 50 ng/mL BDNF for 30 min. SDS-PAGE: 4-12%, reducing conditions; Transfer: Towbin’s Tris-Glycine buffer, PVDF membrane (0.45 μm); Blocking Buffer and antibody diluent: as shown in image, blocking 1 hour at RT; Primary antibody: 1 μg/mL, overnight at 2-8°C; Secondary antibody: anti-rabbit-HRP (1/10,000) 2.5 hours at RT; Detection: Chemiluminiscence.
High skim milk concentration causes suppression of pY817 signal, suggesting that 5% skim milk should be avoided as blocking buffer and diluent. Strongest signal is obtained in 5% BSA blocking buffer, however, many non-specific bands are present. An equal mixture of skim milk and BSA appears to provide the best compromise between signal and noise. However, optimization of blocking condition is recommended for each particular sample, with excess of BSA over skim milk likely to be beneficial for best results.