A) Western Blot analysis of NGF expression in mouse salivary gland homogenate (50 ug, Lane 2) with rabbit polyclonal IgG antibody to beta NGF, R-093-500 (5 µg/mL). R-093-500 detects a strong band at 13 kDa consistent with the expected molecular weight of mature NGF monomer. Lane 1: 100 ng of rhNGF protein.
B) Western Blot analysis of NGF expression in human brain (50 ug, Lane 1) and human PC3 prostate cancer cell lysates (100 ug, Lane 2), with rabbit polyclonal IgG antibody to native NGF, R-093-500 (2 µg/mL). The antibody detects a strong band at 32 kDa consistent with the molecular weight of glycosylated proNGF monomer. ProNGF is known to be the predominant NGF isoform in brain (Fahnenstock et al., 2001). Additional bands are observed at ~22 kDa (non-specific band observed when blotting with pre-immune serum) and ~40 kDa and ~50 kDa. The latter two bands have not been characterized, but might represent differently glycosylated proNGF-isoforms as reported by Reinshagen et al., 2000; Lobos et al., 2005; Pedraza et al., 2005; Pundavela et al., 2014.
Western Blotting Method:SDS-PAGE: denaturing and reducing, 12% Bis-Tris gel; Semi-Dry Transfer: Tris-Glycine (Towbins) buffer with 20% methanol; Membrane: Nitrocellulose (0.45 um); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: 2-5 µg/mL, overnight at 4°C; Secondary antibody: anti-rabbit-HRP (1/6000), 1 hour at RT; Detection: Chemiluminiscence.