Protein isolation Tissue - Mouse liver 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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1 year ago

1 year 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
Whole cell protein was extracted from liver tissue with T-PER tissue protein extraction reagent (Pierce Biotechnology, Rockford, IL, USA). For nuclear protein extraction, the liver samples were collected 60 min after reperfusion and immediately homogenized with a prechilled Dounce homogenizer (Wheaton Industries, Millville, NJ, USA).
Protocol tips
- Cut tissue is resuspended in 3 volumes of CelLytic Buffer plus 10% protease inhibitor mixture and Halt phosphatase inhibitor mixture and incubated on ice for 30.

- After the the livers are lysed using a Qiagen Tissue Lyser LT and then centrifuged at 100,000 × g at 4 °C.
Protocol tips
- For nuclear protein extraction, the liver samples are collected 60 min after reperfusion and immediately homogenized with a prechilled Dounce homogenizer. Nuclear protein is extracted with NE-PER nuclear and cytoplasmic extraction reagents.
Tissue Extraction Reagent I

Thermo Fisher Scientific

Protocol tips
- Total protein was isolated from mouse liver using Tissue Extraction Reagent I supplemented with cOmplete protease inhibitor cocktail.
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