Precautions for protein extraction and purification

Precautions for protein extraction and purification

When purifying any kind of protein, you must always pay attention to maintaining its stability and protecting its activity. There are some general precautions to keep in mind. They include:

1. The operation should be carried out on ice or in a cold storage as far as possible.

2. Don’t be too dilute, and maintain the protein concentration at μg/mL～mg/mL.

3. Suitable pH, unless focusing chromatography, the buffer solution used should avoid the same pH as the pI to prevent protein precipitation.

4. Use protease inhibitors to prevent protease from degrading the target protein; when purifying the protein in the cell, add DNase to degrade the DNA and prevent the contamination of the protein by the DNA.

5. Avoid repeated freezing and thawing and vigorous agitation of the sample to prevent protein denaturation.

6. The components of the buffer solution try to mimic the intracellular environment.

7. Add 0.1~1mmol/LDTT (dithiothreitol) (or β-mercaptoethanol) to the buffer solution to prevent protein oxidation.

8. Add 1~10mmol/LEDTA metal chelating agent to prevent heavy metals from damaging the target protein.

9. Use a sterilizing solution to prevent the growth of microorganisms.

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Protein Isolation and Purification Techniques