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.
Generally it has been difficult to isolate high-quality RNA from yeast because of problems disrupting the cells. Use of enzymes to disrupt cell wall can alter gene expression profiles. Therefore, physical disruption can result in high quality RNA for all downstream processing. Use of DNAse and proteinase K will remove traces of DNA contamination and proteins respectively.
DNA isolation and purification is the first critical step in sample preparation that helps ensure optimal performance of downstream assays like PCR, microarrays, and sequencing. Failure in yielding high-quality DNA would be the major reason that DNA doesn't work for the downstream application. To circumvent this, one should follow the recommended storage conditions to minimize DNA degradation by nucleases and shouldn't overload the purification system.
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