Western blot analysis of proNGF expression in human DU145 prostate cancer cell lysate and normal human brain. Biotinylated, monoclonal proNGF antibody M-1778-50-B (0.5 µg/mL) detects monomeric, non-glycosylated proNGF protein (E.coli derived) at 25 kDa (Lane 1). In DU145 cell lysate (Lane 2) and human brain (Lane 3), M-1778-50-B detects monomeric, glycosylated proNGF at 32 kDa, and several proNGF isoforms (~37 to ~53 kDa). These additional bands have not been characterized, but literature suggests that these bands are at least partly related to glycosylated forms of full-length proNGF (Reinshagen et al., 2000; Lobos et al., 2005; Pedraza et al., 2005; Pundavela et al., 2014). Western Blotting Method: SDS-PAGE: denaturing and reducing, 4-20% Bis-Tris gel; Semi-Dry Transfer: Tris-Glycine (Towbin) buffer with 20% methanol; Membrane: Nitrocellulose (0.45 µm); Blocking: 3% BSA in TBST, 1 hour at RT; Primary antibody: overnight at 4°C; HRP conjugate: streptavidin-HRP (25 ng/mL), 1 hour at RT; Detection: Chemiluminiscence.