Protein isolation Mammalian cells - HeLa

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
The Total exosome RNA and protein isolation kit (Invitrogen) was utilized for recovery of RNA from the exosome samples obtained with the reagent and ultracentrifugation protocol and parental samples for each sample type, HeLa cell pellets (1 × 106 cells) and cell-free serum.
Protocol tips
Cells then were lysed in an appropriate amount of CelLytic™ MT Cell Lysis Reagent (Sigma-Aldrich, C3228) containing 1x protease inhibitor cocktail (Sigma-Aldrich, p8340) on ice with agitation for 30 minutes.
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