1, precipitation.
2. Electrophoresis: protein is charged in a solution above or below its isoelectric point, and can move to the positive or negative pole of the electric field in the electric field. According to the different supports, there are film electrophoresis, gel electrophoresis and so on.
3. Dialysis: the method of separating macromolecular protein from micromolecule compounds with dialysis bags.
4, chromatography:
A, ion exchange chromatography, using protein's amphoteric free property, at a certain PH value, each protein has different charges and properties, so it can be separated by ion exchange chromatography. Such as anion exchange chromatography, protein with small negative charge is eluted first.
B, molecular sieve, also known as gel filtration. Protein, a small molecule, enters the cave and stays for a long time, while protein, a large molecule, cannot enter the cave in time and flows out directly.
5. Ultracentrifugation: It can be used to separate and purify protein and determine the molecular weight of protein. Different protein are distinguished by their different densities and shapes.
Matters needing attention in purifying protein:
1, in order to avoid protein denaturation, do not stir the sample violently and freeze it repeatedly.
2. The composition of the buffer solution simulates the inner ring of cells as much as possible.
3. To avoid the oxidation of protein, add 0. 1 ~ 1 mmol/LDTT (dithiothreitol) (or β-mercaptoethanol) to the buffer solution.
4. In order to avoid the damage of heavy metals to the target protein, 1 ~ 10 mmol/ledta metal chelating agent was added.
5, in order to avoid microbial growth, use sterilization solution. When purifying any kind of protein, we should always pay attention to its stability and protect its activity. We need to remember the following general precautions.
6. Try to operate on ice or in cold storage.
7. protein concentration should not be too thin.
8. Unless it is a focusing layer. Otherwise, the pH value should be appropriate to prevent the pH value of buffer solution from being the same as that of pI, so as to avoid protein precipitation.
9. Use protease inhibitor to avoid the degradation of target protein by protease; In the purification of intracellular protein, dnase is added to degrade DNA to avoid the pollution of DNA to protein.