Pro-nerve growth factor (proNGF), Mouse Monoclonal Antibody

Western blot analysis of proNGF expression in DU145 and PC3 prostate cancer cell lysates. Monoclonal proNGF antibody M-1738-100 detects monomeric, non-glycosylated proNGF protein (20 ng, E.coli derived) at 25 kDa (Sample 1). PC3 (Sample 2, 100 µg) and DU145 (Sample 3, 100 µg) cell lysates show several proNGF isoforms (32 kDa, ~40 kDa, ~60 kDa, and higher molecular weight bands) as previously described, which most likely relates to glycosylated forms of full-length proNGF (Reinshagen et al., 2000; Lobos et al., 2005; Pedraza et al., 2005; Pundavela et al., 2014). Specificity of these bands is shown by pre-incubation with the immunizing peptide (5x mass of primary antibody), and a secondary antibody only control blot. Western Blotting Method: SDS-PAGE: denaturing and reducing, 4-20% Bis-Tris gel; Semi-Dry Transfer: Tris-Glycine (Towbin's) buffer with 20% methanol; Membrane: Nitrocellulose (0.45 µm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: 2 µg/mL, overnight at 4°C; Secondary antibody: anti-mouse-HRP (1/6000), 1 hour at RT; Detection: Chemiluminiscence.

Immunofluorescence analysis of proNGF expression in human DU145 prostate cancer cells. Fixed (4% formaldehyde), permeabilized, and blocked (10% normal horse serum, 0.1% Triton X100) DU145 cells were incubated with proNGF antibody M-1738-100 (2 µg/mL, green) for 1 hour. Primary antibody binding was visualized with a secondary donkey anti-mouse-CF488A antibody (4 µg/mL, 1 hour incubation). Cell nuclei were stained with Hoechst dye (blue). Magnification: 100x.

Fixing and Permeabilization of cells: Absolute methanol (10 minutes in ice) and 0.1% Tween-20 in PBS, Blocking: 200 µg/mL Normal Sheep IgG (20 minutes), Primary antibody: Mouse Monoclonal antibody to proNGF (cat # M-1738-100, 2 ug per ~10^6 cells) for 60 minutes at room temperature, Secondary antibody: Goat anti-mouse PE labeled secondary antibody (1:100 fold dilution) with incubation for 20 minutes in dark at room temperature. Non-specific Control IgG, clone X63 (cat # M-1249-100) was used as negative control under same conditions (black dashed). Flow cytometry data and results were generated using Orflo MoxiflowTM instrument and protocols. The data demonstrates specific staining of proNGF expressed in human prostate cancer DU145 cell line using cat# M-1738-100.

Western blot analysis of proNGF expression in human brain homogenates. Monoclonal proNGF antibody M-1738-100 (2 µg/mL) detects monomeric, non-glycosylated proNGF protein (30 ng, E.coli-derived) at 25 kDa (Sample 1). Brain homogenate 1 (Sample 2, 50 ug) and 2 (Sample 3, 30 ug) show several proNGF isoforms (~53 kDa, ~60 kDa, and ~73 kDa) as previously described, which most likely relates to glycosylated forms of full-length proNGF (Reinshagen et al., 2000; Lobos et al., 2005; Pedraza et al., 2005; Pundavela et al., 2014). Specificity of these bands is shown by pre-incubation with the immunizing peptide (5x mass of primary antibody). Western Blotting Method: SDS-PAGE: denaturing and reducing, 4-20% Bis-Tris gel; Semi-Dry Transfer: Tris-Glycine (Towbin's) buffer with 20% methanol; Membrane: Nitrocellulose (0.45 um); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: overnight at 4°C; Secondary antibody: anti-mouse-HRP (1/6000), 1 hour at RT; Detection: Chemiluminiscence.