Tyrosine Kinase Receptor B, phospho Ser478/479 (TrkB, pS478/479), Rabbit Polyclonal Antibody

Western blot of TrkB (pS478) in mouse NSC34 cell lysates (20 µg/lane). TrkB receptor expression was induced by treating NSC34 cells with retinoic acid. TrkB receptor phosphorylation was triggered by adding BDNF protein to the culture medium, while control cells were left untreated. R-1718-50 (1 µg/mL) detects a band at about ~120 kDa in BDNF-treated cells only, corresponding to full-length TrkB protein phosphorylated at amino acid residue serine 478. Lower molecular weight bands are not characterized, but are likely unrelated to phosphorylation due to equal band intensities in BDNF-treated and untreated cells. SDS-PAGE: denatured and reduced; Transfer: Tris-Glycine buffer; Membrane: nitrocellulose (0.45 µm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: overnight at 2-8°C; Secondary antibody: anti-rabbit-HRP (1/6000) 2 hours at RT; Detection: Chemiluminiscence.

Immunohistochemical analysis of TrkB (pS478) expression in mouse cerebral cortex, including corpus callosum, using rabbit antibody R-1718-50. Mice were perfused (4% PFA), post-fixed followed by cryopreservation with 30% sucrose and frozen on isopentane. Saggittal cryostat sections were blocked with 10% NDS, 1% BSA in 1x TBS with 0.3% Triton-X100 (15 min, room temperature). Sections were incubated with primary antibody R-1718-50 (1:200, red) at 2-8°C overnight, with subsequent application of donkey anti-rabbit-Alexa594 secondary antibody (1:400, 2 hour incubation at room temperature). Sections were co-labelled with goat anti-PDGFRa (green) as positive control. Scale bar = 100 µm. Images courtesy of Dr Jessica Fletcher, University of Melbourne, Australia.

Western blot analysis of TrkB (pS478) in mouse NSC34 RIPA cell lysates and mouse cortex homogenate (20 μg protein/lane). Cells were grown in proliferation medium in presence and absence of Retinoic Acid (RA, 1 μM) for 5 days. Cells were then serum-starved for 4 hours and treated with 50 ng/mL BDNF for 5-30 min. Control experiment: Mouse cortex RIPA homogenate. SDS-PAGE: 4-12%, reducing conditions; Transfer: Towbin’s Tris-Glycine buffer, PVDF membrane (0.45 μm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: 1 μg/mL, overnight at 2-8°C; Secondary antibody: anti-rabbit-HRP (1/10,000) 2 hour at RT; Detection: Chemiluminiscence.

R-1718-50 reveals a single band at ~120 kDa demonstrating detection of pTrkB (S478). RA and BDNF treatment increase band intensity. The membrane was then stripped and re-probed with rabbit antibody to GAPDH (R-1701-100, 1:10,000, following WB procedure as above) to assess well-to-well loading consistency. Densitometric band analysis for GAPDH and pTrkB (S478) shows equal loading amounts per well, confirming increase in TrkB (S478) phosphorylation upon treatment.

In mouse cortex, pTrkB (S478) is detected at ~120 kDa, with additional uncharacterized lower molecular weight bands.

Western blot analysis of TrkB pS478 expression in NSC34 RIPA cell lysates (10 μg protein/lane) and mouse cortex (30 μg protein/lane) with and without lambda-phosphatase treatment. Cells were grown in proliferation medium in presence and absence of Retinoic Acid (RA, 1 μM) for 5 days. Cells were then serum-starved for 4 hours and treated with 50 ng/mL BDNF for 30 min. SDS-PAGE: 4-12%, reducing conditions; Transfer: Towbin’s Tris-Glycine buffer, PVDF membrane (0.45 μm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: 1 μg/mL, overnight at 2-8°C; Secondary antibody: anti-rabbit-HRP (1/10,000) 2 hour at RT; Detection: Chemiluminiscence.

R-1718-50 detects one single strong band at ~120 kDa in NSC34 cell lysates, consistent with pTrkB (S478) expression. Lambda-phosphatase treatment of the membrane obliterates pTrkB (S478) detection, confirming that the detected band is indeed pTrkB (S478). In mouse cortex, lambda-phosphatase treatment causes the disappearance of the pTrkB (S478) band at ~120 kDa, while band intensity for the other 2 uncharacterized lower molecular weight bands remains largely unchanged.

Comparison of blocking buffers for Western blot analysis of TrkB pS478 expression in NSC34 RIPA cell lysates (10 μg protein/lane) and mouse cortex (30 μg protein/lane) using rabbit antibody R-1718-50. SDS-PAGE: 4-12%, reducing conditions; Transfer: Towbin’s Tris-Glycine buffer, PVDF membrane (0.45 μm); Blocking Buffers tested: 5% skim milk in TBST, 5% BSA in TBST, 2.5%/2.5% skim milk/BSA in TBST, 1 hour at RT; Primary antibody: 1 μg/mL, overnight at 2-8°C; Secondary antibody: anti-rabbit-HRP (1/10,000) 1.5 hours at RT; Detection: Chemiluminiscence.

Lowest background is achieved with 5% skim milk blocking buffer. While skim milk appears to slightly reduce signal intensity, it provides cleanest blots. To achieve highest sensitivity with acceptable background, a mixture of skim milk and BSA (each 2.5%) appears to provide the best compromise as blocking buffer for R-1718-50 antibody (TrkB pS478).

Western blot analysis of TrkB pS478 expression in Retinoic Acid (RA)- and BDNF-treated (50 ng/mL BDNF, 15 minutes) NSC34 cells, and untreated rodent tissue homogenates. Samples were lysed with RIPA buffer and 10 μg loaded for cell lysates and 40 μg for tissue homogenates. SDS-PAGE: 4-12%, reducing conditions; Transfer: Towbin’s Tris-Glycine buffer, PVDF membrane (0.45 μm); Blocking buffer and antibody diluent: Mixture of 2.5% skim milk and 2.5% BSA in TBST, blocking 1 hour at RT; Primary antibody: 1 μg/mL, overnight at 2-8°C; Secondary antibody: anti-rabbit-HRP (1/10,000) 2.5 hours at RT; Detection: Chemiluminiscence.

Tissue samples show expression of TrkB phosphorylated at serine residue 478. The RA/BDNF-treated cell lysate serves as positive control supporting the assignment of the phosphorylated TrkB band. Additional bands are observed in tissue samples that have not yet been characterized.