Quantification of a neurotrophin receptor from submilligram quantities of brain tissue using western blotting.
The immunodensity of the trkB neurotrophin receptor was quantified from submilligram quantities of brain tissue, using approximately 500 microgram samples dissected from the cochlear nucleus (CN) of adult guinea pigs. Tissue samples were hand-homogenized in a lysis buffer. After complete lysis, an aliquot of the lysate was taken to measure total protein, and the remainder was denatured. Proteins in the denatured aliquot were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto an immoblin-P membrane. The membrane was exposed to rabbit anti-trkB and then to anti-rabbit IgG HRP conjugate. The immuno-complex on the membrane was detected by chemiluminescence, which was recorded on autoradiographic film. Autoradiographs were scanned into a computer and the trkB immunobands were quantified by densitometry. This procedure allowed the quantitative comparison of trkB neurotrophin receptor immunodensities between tissue samples. The procedure can be applied to the analysis of small samples of tissue collected from different brain regions or collected from the same region at different times, such as during development or aging, after administering a drug, or after placing a lesion.
Suneja
S K
SK
Department of Anatomy, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3405, USA. ssuneja@neuron.uchc.edu
Potashner
S J
SJ
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D017626:Cochlear Nucleus / Q000737:chemistry*; D005260:Female; D006168:Guinea Pigs; D008297:Male; D008832:Microchemistry; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis*; D020801:Receptor, Ciliary Neurotrophic Factor; D017475:Receptors, Nerve Growth Factor / Q000032:analysis*
D020801:Receptor, Ciliary Neurotrophic Factor; D017475:Receptors, Nerve Growth Factor; D020794:Receptor Protein-Tyrosine Kinases