Western blotting was performed as previously described [17 (link)]. In brief, protein concentrations of samples were determined using the BCA protein assay (Bio-Rad, Hemel Hempstead, Herts, UK). Twenty micrograms of each sample were subjected to Western blot analysis using the following primary antibodies: rabbit polyclonal anti-phospho-tyrosine receptor kinase B (TrkB) (phospho Y515, abeam, Cambridge, MA, USA) at 1:1000 dilution, rabbit polyclonal anti-TrkB (abeam) at 1:1000 dilution and rabbit polyclonal anti-α-tubulin (cell signaling Technology Inc.) at 1:1000 dilution. Images were scanned by an Image Master II scanner (GE Healthcare, Milwaukee, WI, USA) and analyzed using ImageQuant TL software v2003.03 (GE Healthcare). The band signals of the interesting proteins were normalized to those of the corresponding α-tubulin and expressed as fractions of control sample from the same gels.
Protocol detail
Western Blot Analysis of Phospho-TrkB
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Antibodies
Assay
Dilution
Gels
Hemel
Proteins
Rabbit
Tyrosine receptor kinase
Western blot analysis
α tubulin
Corresponding Organization :
Other organizations : University of Virginia
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Variable analysis
independent variables
- None explicitly mentioned
- Phosphorylation levels of tyrosine receptor kinase B (TrkB) protein
- Relative abundance of TrkB protein
- Protein concentration of samples determined using BCA assay
- Loading of 20 μg of each sample for Western blot analysis
- Use of anti-α-tubulin antibody as a loading control
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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