Let’s suppose you want to sequence a protein.
Well, the first step is to ensure that your protein is kept stable. Factors that are important to protein stability include:
- pH – If a protein is not kept in its natural pH state, it will denature, resulting in structural disruption (or chemical degradation). That’s why scientists use buffer solutions to make sure the protein is kept in an environment that keeps the protein in the proper pH environment.
- Temperature also affects protein stability so it is important to know the temperature limits of the protein you are working with. Some proteins can denature at just a few degrees higher than its native state. However, most protein purification is carried out at temperatures near 0°C
- Presence of degradative enzyme – When you destroy other tissue to get the protein sample you wish to work with, degradative enzymes can be released such as proteases (an enzyme that breaks down proteins and peptides) and nucleases (enzymes that cleave nucleotides). The presence of degradative enzymes can be dealt with by adjusting ph or temperature to prevent the degradative enzymes from functioning, the inhibiting protein degradation.
- Adsorption to surfaces – Many proteins can denature while coming in contact with air-water environments or with glass and plastic surfaces. Thus, proper solutions are created to preserve the protein.
- Long-term protein storage – Proteins can degrade if stored for a long time because microbial contamination and slow oxidation can completely destroy your protein. Nitrogen gas is sometimes employed to preserve proteins for long-term use.
Protein Purification Methods:
The goal of protein purification is to eliminate other components of a protein mixture so that only the desired protein remains.
Here are a few of the common methods employed:
- Salting out proteins based on solubility
- Ion exchange chromatography
- Hydrophobic interaction chromatography
- Gel filtration chromatography