Protein isolation Mammalian cells - THP-1

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
- The supernatants are precipitated with 10% trichloroacetic acid. For concentration by desalinization, washed with acetone, and then dried at room temperature. The dried pellets and the remaining cells were suspended in a reducing sample buffer containing 2-mercaptoethanol.
RIPA Buffer

Sigma-Aldrich

Protocol tips
- Extraction of protein from cells was performed with radioimmunoprecipitation assay buffer lysis buffer according to manufacturer's protocol.
Cell Lysis Buffer (10X)

Cell Signaling Technology

Protocol tips
Proteins were extracted from cultured cells using lysis buffer (Cells Signaling Technology, Danvers, MA, United States).
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