(1) Separation method based on ligand specificity-affinity chromatography
Affinity chromatography (aflinitychromatography) is an extremely effective method for protein separation. It often requires only one step to separate a certain protein to be purified from a very complex protein mixture, and the purity is very high. This method is based on the fact that certain proteins can bind specifically to another molecule called a ligand (Ligand) rather than covalently. The basic principle: Proteins exist in the form of complex mixtures in tissues or cells. Each type of cell contains thousands of different proteins. Therefore, protein separation, purification, and characterization are An important part of biochemistry, there is no single or a set of ready-made methods to remove any kind of protein from a complex mixed protein, so it is often used in combination.
(2) Separation method according to the difference of protein molecule size
1. Dialysis and ultrafiltration
Dialysis uses semi-permeable membranes to separate proteins with different molecular sizes.
The ultrafiltration method uses high pressure or centrifugal force to force water and other small solute molecules to pass through the semi-permeable membrane, while the protein is left on the membrane. A lubricating membrane with different pore sizes can be selected to retain proteins of different molecular weights.
2. Gel filtration method
Also known as size exclusion chromatography or molecular sieve chromatography, this is one of the most effective methods for separating protein mixtures based on molecular size. The most commonly used packing materials in columns are glucose gel (Sephadex ged) and agarose gel.
(3) Separation according to the charged properties of proteins
Proteins have different charging properties and different amounts of charge in different pH environments, which can be separated.
1. Electrophoresis
Under the same pH condition, various proteins can be separated due to different mobility in the electric field due to different molecular weight and charge quantity. It is worth noting that isoelectric focusing electrophoresis, which uses a kind of ampholyte as a carrier, the ampholyte forms a pH gradient that gradually increases from the positive electrode to the negative electrode during electrophoresis. When a certain charged protein swims in it, it reaches the respective The pH position of the electric point stops, and this method can be used to analyze and prepare various proteins.
2. Ion exchange chromatography
Ion exchangers are cation exchangers (eg: carboxymethyl cellulose; CM-cellulose) and anion exchangers (diethylaminoethyl cellulose; cellulose), when the separated protein solution flows through ion exchange chromatography During the column, the protein with the opposite charge to the ion exchanger is adsorbed on the ion exchanger, and then the adsorbed protein is eluted by changing the pH or ionic strength.
(4) Separation methods according to different protein solubility
1. Salting out of protein
Neutral salt has a significant effect on the solubility of protein. Generally, the solubility of protein increases with the increase of salt concentration at low salt concentration. This is called salt dissolution; when the salt concentration continues to increase, the solubility of protein decreases to different degrees and successively Precipitation. This phenomenon is called salting out. A large amount of salt is added to the protein solution. The high concentration of salt ions (such as SO4 and NH4 of ammonium sulfate) has a strong hydration power, which can seize the hydration layer of protein molecules, so that The "water loss", the protein micelles coagulate and precipitate out. The effect is better if the pH of the solution is at the isoelectric point of the protein during salting out. Due to the different particle sizes and hydrophilicity of various protein molecules, the salt concentration required for salting out is also different. Therefore, the neutral salt concentration in the mixed protein solution can be adjusted to precipitate various proteins in stages.
The factors that affect salting out are: (1) Temperature: In addition to temperature-sensitive proteins operating at low temperatures (4 degrees), they can generally be performed at room temperature. Generally, the protein solubility decreases at low temperatures. However, some proteins (such as hemoglobin, myoglobin, albumin) are more soluble at higher temperatures (25 degrees) than at 0 degrees and are more likely to salt out. (2) pH: Most proteins have the lowest solubility in concentrated salt solution at the isoelectric point. (3) Protein concentration: When the protein concentration is high, the protein to be separated is often precipitated together with other proteins (coprecipitation). Therefore, before the salting out, the serum should be diluted with the same amount of normal saline, so that the protein content is 2.5-3.0%.
Commonly used neutral salts for protein salting out are mainly ammonium sulfate, magnesium sulfate, sodium sulfate, sodium chloride, sodium phosphate, etc. Among them, the most used ammonium sulfate has the advantages of small temperature coefficient and high solubility (saturated solution at 25 degrees is 4.1M, which is 767 grams / liter; at 0 degrees, the saturated solubility is 3.9M, which is 676 grams / liter). Within this solubility range, many proteins and enzymes can be salted out; in addition, the ammonium sulfate staged salting out effect is also better than other salts, and it is not easy to cause protein denaturation. The pH of the ammonium sulfate solution is usually between 4.5 and 5.5. When salting out with other pH values, sulfuric acid or ammonia water is required.
After the protein is separated by salting-out precipitation, the salt in the protein needs to be removed. The common method is dialysis, that is, the protein solution is put into the Xiu analysis bag (usually cellophane), and the buffer is used for dialysis, and it is continuously replaced. The buffer solution requires a long time for dialysis, so it is best to perform it at a low temperature. In addition, G-25 or G-50 can be used to remove salt through the column, the time is shorter
2. Isoelectric precipitation method
When the protein is in the electrostatic state, the electrostatic repulsion between the particles is the smallest, so the solubility is also the smallest. The isoelectric point of various proteins is different. The pH of a solution can be adjusted to reach the isoelectric point of a protein to precipitate it, but this method Less used alone, can be combined with salting out method.
3. Low temperature organic solvent precipitation method
Using water-miscible organic solvents, methanol, ethanol or acetone, can reduce the solubility of most proteins and precipitate out. This method has higher resolution than salting out, but the protein is more volatile and should be carried out at low temperature.
Zhejiang Shengfa Textiles Printing&Dyeing.,ltd , https://www.chzjsf.com