Immunofluorescence analysis of proBDNF expression in human SHSY-5Y cells. Fixed (4% formaldehyde), permeabilized, and blocked (10% normal horse serum, 0.1% Triton X100) SHSY-5Y cells were incubated with proBDNF antibody S-079-100 (1:1000, red) for 1 hour. Primary antibody binding was visualized with a secondary donkey anti-sheep-PE antibody (1:100, 1 hour incubation). Cell nuclei were stained with Hoechst dye (blue). BDNF-IR is observed in the perinuclear cytoplasm. Magnification: 100x.
Separation and detection of proBDNF in conditioned 293 cell culture medium (Lanes 1 and 3, 50 ng/lane) and purified mature BDNF (Lanes 2 and 4, 50 ng/lane) by reduced SDS-PAGE and western blotting. Anti-proBDNF sheep antibody S-079-100 (1/1000) detects 2 bands (Lane 1) with the apparent molecular weight of ≈ 32 kDa (red arrow) and ≈ 64 kDa (blue arrow) corresponding to glycosylated proBDNF monomer and dimer, respectively. S-079-100 is specific for proBDNF and does not recognize mature BDNF (Lane 2). Rabbit anti-BDNF antibody R-1707-100 (0.2 µg/mL) is a superb antibody for western blotting. It detects mature BDNF (≈ 14 kDa, Lane 4, black arrow) and shows lower affinity for pro-BDNF monomer (Lane 3, red arrow) and proBDNF dimer (Lane 3, blue arrow).