NGFR/p75 neurotrophin receptor (p75NTR), Mouse Monoclonal Antibody

Left: Analysis of p75NTR expression in human and rodent RIPA cell lysates by Western Blotting. Mouse antibody to p75NTR clone 8J2 (M-1818-100) and clone MLR2 (M-009-100) detect p75NTR-IR at ~50-60 kDa in mouse p75NTR-transfected cells (A), but not in non-transfected control cells (B). p75NTR-IR is also observed in human SH-SY5Y (C) and rat C6 (D) cell lysates. Dependent on cell lysate, dimers or trimers (~150 kDa) of p75NTR are detected (Anastasia et al., 2015). 50 ug of protein were loaded per lane. WB Method: SDS-PAGE: 4-12%, non-reducing conditions; Transfer: Tris-Glycine buffer; Membrane: nitrocellulose (0.45 um); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: 1 µg/mL, overnight at 4°C; Secondary antibody: anti-mouse-HRP (1/6000) 1 hour at RT; Detection: Chemiluminiscence. Right: Immunoprecipitation of p75NTR from rat C6 cell lysate and detection by Western Blotting. Mouse antibody to p75NTR clone 8J2 (M-1818-100) and clone MLR2 (M-009-100) precipitate bands at the expected molecular weight of ~50-60 kDa for p75NTR (A). No p75NTR-IR is observed in remaining supernatant (B) or control (Protein G only, C). IP Method: C6 lysates were prepared in non-denaturing lysis buffer and pre-cleared with Protein G agarose beads (40 uL bead slurry per 1 mg total protein). Cleared lysate was then incubated with either M-1818-100 or M-009-100 (10 ug antibody per 200 ug total protein), and immune complexes bound by Protein G agarose (10 uL). Precipitated p75NTR was eluted off the beads and antibody by heating and addition of SDS-PAGE sample buffer. WB Method: as above, with primary goat anti-p75NTR (1 µg/mL) antibody and secondary anti-sheep-HRP (1/6000) antibody. Detection: Chemiluminiscence.

Binding analysis of M-1818-100 (8J2) and M-009-100 (MLR2) antibodies by 1-site ELISA on human and mouse p75NTR protein. Clone 8J2 detects both, human and mouse p75NTR proteins with higher affinity than clone MLR2.

Analysis of p75NTR expression in human SH-SY5Y and rat C6 cell lines by Flow Cytometry. Blocking: 200 µg/mL sheep IgG, 30 minutes on ice; Primary antibody: Mouse monoclonal antibodies to p75NTR cat # M-1818-100, clone 8J2 (red) and M-009-100, clone MLR2 (black), ~2 µg per 10^6 cells, for 60 minutes on ice, Secondary antibody: Goat anti-mouse-PE (1:100 dilution), 20 minutes in dark on ice. Negative control: Non-specific Control IgG, clone X63 (cat # M-1249-200, black dashed). Data and results were generated using Orflo Moxiflow^TM instrument and protocols.

Analysis of membrane p75NTR expression in rat C6 cells by Immunocytochemistry. Non-permeabilized cells were stained with mouse antibodies to p75NTR clones 8J2 (M-1818-100) and MLR2 (M-009-100) either on ice without fixation (top panel), or after fixation with 4% formaldehyde (bottom panel). Both antibodies reveal punctuate membrane staining of p75NTR receptor. This staining is specific, because negative mouse control antibody clone X63 (M-1249-100) does not show immunoreactivity. Method: Primary antibodies: 2 µg/mL, 1 hour incubation; Blocking: 10% normal horse serum, 30 minutes; Fixation: 4% formaldehyde, 10 minutes, either before application of primary antibody (bottom panel) or after application of primary antibody (upper panel); Secondary antibody: donkey anti-mouse-CF488A (2 µg/mL), 1 hour incubation. Cell nuclei were stained with Hoechst dye (blue), 1:1,000, 20 minutes at room temperature. Magnification: 100x.

p75NTR staining (green) in rat trigeminal ganglion cells by Immunocytochemistry. Primary antibody dilution: 1:100. Image courtesy of QBM Cell Science.