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Methods and precautions for protein purification

Update time : 2020-10-26

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Methods and precautions for protein purification
The first step in protein research is to separate and purify the target protein from a complex mixture of macromolecules to obtain a high-purity target with biological activity. Therefore, efficient protein purification techniques and methods are one of the important foundations and keys of protein research.

Protein extraction and purification method

Extraction of protein (including enzymes)

Most proteins are soluble in water, dilute salt, dilute acid or alkali solutions, and a few proteins bound to lipids are soluble in organic solvents such as ethanol, acetone, butanol, etc. Therefore, different solvents can be used for extraction, separation and purification. Protein and enzymes.

(1) Aqueous solution extraction method

The aqueous solution of dilute salt and buffer system has good stability and high solubility for protein. It is the most commonly used solvent for protein extraction. 

The usual amount is 1-5 times the volume of the raw material. The extraction requires uniform stirring to facilitate protein dissolution. The extraction temperature depends on the nature of the active ingredients.


On the one hand, the solubility of most proteins increases as the temperature rises. Therefore, higher temperatures are conducive to dissolution and shorten the extraction time.

But on the other hand, the increase in temperature will denature and inactivate the protein. Therefore, based on this point, the extraction of proteins and enzymes generally uses low temperature (below 5 degrees) operation. 

In order to avoid degradation during protein extraction, proteolytic enzyme inhibitors (such as diisopropyl fluorophosphate, iodoacetic acid, etc.) can be added.



(2) Organic solvent extraction method

Some proteins and enzymes that are more firmly bound to lipids or have more non-polar side chains in the molecule are insoluble in water, dilute salt solutions, dilute acids or dilute alkalis. 

Organic solvents such as ethanol, acetone and butanol can be used. It has certain hydrophilicity and strong lipophilicity. It is an ideal lipoprotein extraction solution. But it must be operated at low temperatures.


The butanol extraction method is particularly superior for extracting some proteins and enzymes that are tightly bound to lipids. One is that butanol has strong lipophilicity, especially its ability to dissolve phospholipids; the other is that butanol has both hydrophilicity and is within the solubility range. 

(Degree is 10%, 40 degree is 6.6%) will not cause enzyme denaturation and inactivation. In addition, the butanol extraction method has a wide range of pH and temperature options, and it is also suitable for animal, plant and microbial materials.


Precautions for protein extraction and purification:


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

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

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

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

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

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

6. The components of the buffer solution mimic the intracellular environment as much as possible.

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 sterilizing solution to prevent the growth of microorganisms.

GeneralBiosystems can provide customized protein expression and purification services to meet most of your protein production needs.