Protein isolation Tissue - Human aortic endothelial cells

Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.

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4 years ago

4 years ago by Jesualdo Hernández Spain

When should I be harvesting the tissue before using it for the most optimal results?

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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Protocol tips
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.
Protocol tips
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).
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