Protein isolation Mammalian cells - Caco-2

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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Found 3 matching solutions for this experiment

Protocol tips
- Instructions are followed regarding the manufacturer protocol, except fractions corresponding to cytosol, membrane and nuclei were boiled 5 min at 95°C in the presence of Laemmlli sample buffer.
CelLytic™ M

Sigma-Aldrich

Protocol tips
- Cells are lysed using CelLytic MT mammalian tissue lysis/extraction reagent containing 1% complete protease inhibitor mixture according to the manufacturer’s protocol.
Protocol tips
After cell harvesting, the pellets were washed three times with cold PBS, lysed with RIPA buffer containing protease inhibitors, and sonicated using a sonicator
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