I want to work on in vitro axonal degeneration caused by prion peptide 106-126.
I want to confirm the best possible pathway of degeneration via western blot analysis. Can anyone help please?
Defects in axonal transport and synaptic dysfunctions are associated with early stages of several neurodegenerative diseases including Alzheimer’s, Huntington’s, Parkinson’s, and prion diseases. You should make peptide structure known with its folds and amino acids,. Maintain neuronal M2 cell culture, for treatment and do cytotoxicity assay. Take observations under an inverted microscope (Nikon Eclipse TE2000-U, Nikon, Melville, NY, USA) with an illumination system X-Cite 120 connect through fiber-optics using a 1.3 aperture Plan Fluor 100x numerical aperture and 20x/0.50 numerical aperture objectives, Apply immune staining, cytotoxicity assay, The level of cellular isoform of the prion protein (PrP C ) expression will increasses as differentiation of NTERA2 (NT2) cells pro-gresses. (a) perform Western blot analysis for finding expression of type III b -tubulin (top panel) and PrP C (bottom panel) in NT2 cells at different stages of incubation with retinoic acid (RA). PrP C should stained by using 3F4 anti- body. The amount of total protein loaded into sodium dodecyl sulfate– polyacrylamide gel electrophoresis will be estimated by bicinchoninic acid assay protein reagent and normalized for each sample taken at different time points. Staining for actin with monoclonal antibody against b-actin (1 : 5000, Sigma) will be used as a loading control. (b) Terminally differentiated NT2 cells stained for PrP C (left panel) and for the neuronal marker GAP43 (right panel). Combined image (bottom panel) shows that PrP C was not only expressed in NT2/N (GAP43-positive) cells, but also in NT2/G cells. PrP C will be detected using 3F4 antibody.
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