Protein isolation Tissue - Mouse lung tissue

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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4 years ago

4 years ago by Jesualdo Hernández Spain

When should I be harvesting the tissue before using it for the most optimal results?

I am planning to do HIF nuclear extraction using mouse brain tissue. How fast should I be harvesting the tissue before using it for the most optimal results?

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Protocol tips
- T-PER tissue extraction reagent containing Halt protease inhibitor mixture (Pierce Technology) at a final concentration of 1× is added at 20 ml/g of tissue, and samples are homogenized on ice. Tissue debris is removed by centrifugation for 5 min at 10,000 rpm.
Cell Lysis Buffer (10X)

Cell Signaling Technology

Protocol tips
- The tissue is lysed in cell lysis buffer (Cell Signaling) supplemented with complete EDTA-free protease inhibitors, PhosSTOP phosphatase inhibitors, 1 mM PMSF and 50 mM NaF.
Protocol tips
- The tissues are homogenised in CelLytic MT Cell Lysis Reagent (Sigma C3228), protease inhibitors, phosphatase inhibitors and vanadate and centrifuged at 4°C.
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