Western Blot analysis of NGF expression in rodent and human homogenates. Mature NGF is detected in mouse salivary gland homogenate at ~13 kDa, while tissue samples reveal predominantly proNGF isoforms at higher molecular weights. This is consistent with the observation that proNGF is the predominant isoform in brain tissue (Fahnenstock et al., 2001). Several proNGF-bands are observed, likely representing differently glycosylated proNGF-isoforms as reported by Reinshagen et al., 2000, Lobos et al., 2005, Pedraza et al., 2005 and Pundavela et al., 2014. Incubation of membrane with pre-immune serum reveals some non-specific bands at ~40 kDa, while overall confirming NGF-immunoreactivity.
Western Blotting Method: SDS-PAGE: denaturing and reducing, 10% Bis-Tris gel; Semi-Dry Transfer: Tris-Glycine (Towbins) buffer with 20% methanol; Membrane: PVDF (0.45 µm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: 5 µg/mL, overnight at 2-8°C; Secondary antibody: anti-rabbit-HRP (1/20,000), 2 hours at RT; Detection: Chemiluminiscence.