Western blot analysis of BDNF expression in acid-treated rodent and human brain samples. Brain samples were acid-treated at pH 4 (refer to References for a detailed extraction method protocol). M-1744-50/100 (1 µg/mL) detects BDNF monomer at 14 kDa. Additional higher MW bands are not characterized, but may be related to the cross-reaction of the secondary anti-mouse-HRP antibody with endogenous, reduced IgG present in samples, in particular mouse tissue.
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-mouse-HRP (1/6000), 2 hours at RT; Detection: Chemiluminiscence.