Analysis of membrane TrkB expression in three human cell lines by Flow Cytometry. Staining was perfomed under native conditions. Blocking: 10% normal horse serum; Primary antibody: R-1834-100 (red), 10 µg/mL, 30 minutes on ice; Secondary antibody: donkey anti-rabbit PE (1:100), 30 minutes in dark on ice. Negative control: normal rabbit IgG (black). Data and results were generated using Orflo MoxiflowTM instrument and protocols.
Western Blot and Immunoprecipitation analysis of TrkB expression in cell lysate and tissue homogenates. Rabbit anti-TrkB antibody detects two main TrkB isoforms at ~140 kDa (full-length) and ~100 kDa (truncated TrkB, TrkB-T1) in tissue homogenates (red arrows). In cell lysate, a TrkB isoform at ~50 kDa is observed (black arrow) which is immunoprecipitated with mouse anti-TrkB clone BS379 (M-1836-100). Additional uncharacterized bands are observed. Western Blotting Method: SDS-PAGE: denaturing and reducing, 4-12% Bis-Tris gel; Transfer: Towbin’s transfer buffer; Membrane: PVDF (0.45 µm); Blocking: 5% skim milk in TBS pH 7.6 (1 hour at RT); Primary antibody: rabbit anti-TrkB (0.5 µg/mL), overnight at 2-8°C; Secondary antibody: anti-rabbit-HRP (1/10,000), 1 hour at RT; Detection: ECL. Immunoprecipitation Method: 2 mg protein lysate in non-denaturing buffer (20 mM Tris, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA, protease inhibitor cocktail) was pre-cleared with Protein G agarose (80 uL of 50% bead slurry) and TrkB precipitated from 500 ug protein lysate with 20 µg TrkB antibody clone BS379 (M-1836-100), or negative control IgG clone X63 (M-1249-100). Immunocomplexes were captured with Protein G agarose (10 uL of 50% bead slurry) and eluted with 25 µL of 4x sample buffer. Precipitated proteins were subjected to Western Blotting as outlined above.
Western Blot analysis of TrkB expression in RIPA cell lysates. Rabbit anti-TrkB antibody detects one TrkB isoforms at ~50 kDa which is immunoprecipitated with mouse antibody to TrkB (M-1836-100). In all cell lysates tested, R-1834-100 does not detect ~140 kDa TrkB (full-length) or ~100 kDa (truncated TrkB, TrkB-T1). Western Blotting Method: SDS-PAGE: denaturing and reducing, 10% Bis-Tris gel; Transfer: Towbin’s Transfer buffer; Membrane: PVDF (0.45 µm); Blocking: 5% skim milk in TBS pH 7.6 (1 hour at RT); Primary antibody: rabbit anti-TrkB (0.5 µg/mL), overnight at 2-8°C; Secondary antibody: anti-rabbit-HRP (1/10,000), 1 hour at RT; Detection: ECL.