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2 years ago
2 years ago by Jesualdo Hernández
I am planning to do HIF nuclear extraction using mouse brain tissue. How fast should I be harvesting the tissue before using it for the most optimal results?
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|Brain tissue was lysed in Mammalian Protein Extraction Reagent (M-PER) with phosphatase and protease inhibitors (10 μl/ml, Halt™ Protease and Phosphatase Inhibitor Cocktail, Thermo Scientific Inc., Rockford, IL, USA) using sonication, left to incubate on ice for 10 min and centrifuged at 20,000g at 4 °C for 20 min. The supernatant was taken, and centrifuged at 9000g at 4 °C for 5 min. Supernatant was taken again and used for further analysis.|
|To extract membrane proteins and cell surface proteins, cell lysates were prepared and processed from proliferating (PDL23) and senescent (PDL59) WI-38 fibroblasts using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Scientific) or the Pierce™ Cell Surface Protein Isolation Kit (Thermo Scientific).|