Protein isolation Mammalian cells - Human lung fibroblasts

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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Protocol tips
Total cellular protein was prepared from fibroblasts with 1% IGEPAL lysis buffer supplemented with a protease inhibitor cocktail (leupeptin, aprotinin, pepstatin, and PMSF; Sigma). Cell lysates were centrifuged (14,000 g, 4°C for 10 min) to remove debris, and protein quantitation was performed with the bicinchoninic acid (BCA) method according to the manufacturer's instructions (Pierce, Rockford, IL).
RIPA Buffer


Protocol tips
Cells were washed twice with ice cold PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Sigma). Protein content was measured with BCA reagent (Pierce).
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