Protein isolation Mammalian cells - HLE-B3

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
For Western blot analysis, nuclear and cytoplasmic fractions were generated using the CelLytic NuCLEAR Extraction kit (Cat#NXTRACT Sigma-Aldrich,). 48 hours after transfection, cells were washed with PBS, collected, and resuspended in 0.5 ml of hypotonic lysis buffer and incubated on ice for 15 minutes, 30 μl of 10% IGEPAL was added to the cell suspension. Cells were then vortexed and spun at 10,000 xg for 30 seconds at 4°C.
Protocol tips
- Total protein is extracted in protein lysis buffer M-PER. Protein extracts (20 to 40 μg) are denatured in Laemmli's sample buffer, followed by boiling for 5 minutes, and then resolved on a 4 to 20% Tris-glycine gel.
Protocol tips
- Follow the manufacturer's protocol.
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