Quantifying Protein Expression in Brain

Brain tissues were homogenized in RIPA lysis buffer (Boston Bioproducts, Ashland, MA) containing protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO). Western blot analysis was performed as we have described previously [39 (link)]. Briefly, 30 μg of total protein was separated by SDS-PAGE and probed with respective antibodies, 1:1000 for phospho/total AKT, phospho/total ERK1/2 (Cell Signaling, Danvers, MA) followed by incubation with secondary antibody conjugated to horseradish peroxidase for 1 hour. Signals were developed with chemi-luminescence using a super signal ECL kit (ThermoFisher Scientific, Madison WI). Total AKT and ERK1/2 were used as loading controls. Images were obtained using an Odyssey imaging system (LI-COR Biotechnology, Lincoln, NE).

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Wesley U.V., Hatcher J.F., Ayvaci E.R., Klemp A, & Dempsey R.J. (2016). Regulation of dipeptidyl peptidase IV in the post-stroke rat brain and In vitro ischemia: Implications for chemokine mediated neural progenitor cell migration and angiogenesis. Molecular neurobiology, 54(7), 4973-4985.

Publication 2016

RIPA lysis buffer protease inhibitor cocktail phospho/total AKT super signal ECL kit Odyssey imaging system

Antibodies Antibody Brain Buffer Erk1 Horseradish peroxidase Luminescence Protease inhibitor Protein Ripa Sds page Tissues Western blot analysis

Corresponding Organization : Neurological Surgery

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Variable analysis

independent variables

RIPA lysis buffer containing protease inhibitor cocktail

dependent variables

Phosphorylation levels of AKT and ERK1/2

control variables

Total AKT and ERK1/2 levels used as loading controls

controls

No positive or negative controls were explicitly mentioned in the provided information.

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