Western blot analysis of BDNF expression in SH-SY5Y cell lysate. Polyclonal rabbit BDNF antibody to BDNF (129-138; here whole serum antibody R-083-100 at 1:1000 dilution is shown) detects monomeric BDNF at 14 kDa and monomeric proBDNF at 32 kDa. An additional 28 kDa band is prominent which might relate to BDNF dimer or truncated BDNF isoform (Tongiorgi et al., 2012). Western Blotting Method: SDS-PAGE: denaturing and reducing, 12% Bis-Tris gel; Transfer: Tris-Glycine buffer, semi-dry transfer; Membrane: nitrocellulose (0.22 um); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: overnight at 4C; Secondary antibody: anti-rabbit-HRP (1/6000), 2 hours at RT; Detection: Chemiluminiscence.
Western blot analysis of BDNF expression in human serum (A) and human brain (B). Polyclonal rabbit BDNF antibody to BDNF (129-138; here whole serum antibody R-083-100 at 1:1000 dilution is shown) detects monomeric BDNF at 14 kDa in both samples, as well as monomeric proBDNF at 32 kDa in human brain (B). In addition, a 18 kDa BDNF isoform is detected in human serum (A), confirmed with a monoclonal mouse antibody to rhBDNF, Clone 4C8 (M-1744-50/100, 1 µg/mL). A 18 kDa BDNF-isoform has previously been detected with other BDNF antibodies ( Tongiorgi et al., 2012 ; Silhol et al., 2007 ). In human brain (B), a 28 kDa band is prominent which might relate to BDNF dimer or truncated BDNF isoform ( Tongiorgi et al., 2012 ). Western Blotting Method: SDS-PAGE: denaturing and reducing, 12% Bis-Tris gel; Transfer: Tris-Glycine buffer, semi-dry transfer; Membrane: nitrocellulose (0.22 um); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: overnight at 4C; Secondary antibody: anti-mouse-HRP or anti-rabbit-HRP (1/6000), 2 hours at RT; Detection: Chemiluminiscence.