Western blot analysis of BDNF expression in SH-SY5Y cell lysate (RIPA) and human brain. Polyclonal rabbit antibodies to rhBDNF R-088-100 (whole serum, 1:1000) and R-017-500 (IgG, 10 µg/mL) detect monomeric BDNF at 14 kDa in human brain (Tris-homogenate). ProBDNF is detected at the expected molecular weight of 32 kDa for glycosylated proBDNF monomer. A 18 kDa BDNF-isoform is shown that has previously been detected with other BDNF antibodies (Tongiorgi et al., 2012; Silhol et al., 2017). This 18 kDa band is also visible in the proBDNF protein sample, Lane 2.
Western Blotting Method: SDS-PAGE: denaturing and reducing, 12% Bis-Tris gel; Transfer: Tris-Glycine buffer, semi-dry transfer; Membrane: nitrocellulose (0.22 µm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: overnight at 4°C; Secondary antibody: anti-rabbit-HRP (1/6000), 2 hours at RT; Detection: Chemiluminiscence.