Protein isolation Mammalian cells - HEK293T

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

Upstream tips
- The cells were resuspended in RIPA buffer and lysed by sonication at 40% power with four 10-second pulses and a 20 second rest on ice.
Protocol tips
- For nuclear and cytoplasmic protein isolation, the Thermo Fisher NE-PER kit was used according to the manufacturer’s instructions with the exception of isolating nuclear proteins with sonication at 40% power with two 10-second pulses and 20 second rest on ice.
RIPA Lysis and Extraction Buffer

Thermo Fisher Scientific

Protocol tips
Cells were washed with ice-cold PBS and lysed in ice-cold radioimmunoprecipitation assay lysis buffer (Pierce), with added protease inhibitor (Sigma).
CelLytic™ M

Sigma-Aldrich

Protocol tips
- Protocol was performed following the manufacturer's instructions.
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