Protein isolation Tissue - Rabbit eye retina/choroids

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
The other part of the cornea was used for protein extraction. The corneas were weighed and separately chopped into small pieces. The tissue was solidified by immersion in liquid nitrogen and then ground into a powder. This step was repeated three times. Tissue extraction was performed according to the reagent instructions (Tissue Protein Extraction Cocktail, ThermoFisher Scientific, Inc., Waltham, MA, USA).
Protocol tips
Tissue proteins were extracted from the lysates of homogenized retina/choroids using lysis reagent (CellLytic MT, C3228; Sigma-Aldrich Corp., St. Louis, MO, USA) and a homogenizer.
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