(1) Separation method based on ligand specificity-affinity chromatography
Affinity chromatography (aflinity chromatography) separation of proteins is a highly effective method, it is often only after some further processing to be purified so that the protein isolated from complex protein mixtures, and the high purity . This approach is based on the specific, but not covalent, binding of certain proteins to another molecule called Ligand . The basic principle: the protein in the tissue or cell is present in a complex mixture of each type of cell contains thousands of different proteins, thus separating proteins (Separation), purification (Purification) and identification (Characterization) is An important part of biochemistry, a single or a set of off-the-shelf methods that have not yet been removed, can extract any protein from complex mixed proteins, so several methods are often used in combination.
(2) Separation method based on the difference in protein molecular size
1 , dialysis and ultrafiltration
The dialysis method uses a semi-permeable membrane to separate proteins of different molecular sizes.
Ultrafiltration is the use of high pressure or centrifugal force to force water and other small solute molecules to pass through the semi-permeable membrane, while the protein remains on the membrane. The membranes with different pore sizes can be selected to retain proteins of different molecular weights.
2 , gel filtration
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. Column is the most commonly used filler Sephadex (Sephadex ged) and agarose gel (agarose gel).
(3) Separation based on the charged properties of the protein
Proteins can be separated by different charging properties and amounts of charge in different pH environments.
1 , electrophoresis
Various proteins are separated under the same pH conditions due to the difference in molecular weight and the amount of charge in the electric field. It is worthy of attention that isoelectric focusing electrophoresis uses an ampholyte as a carrier. During electrophoresis, the ampholyte forms a pH gradient that gradually increases from positive to negative . When a certain charged protein moves in it, it reaches its own. The pH position of the electrical point is stopped and this method can be used to analyze and prepare various proteins.
2 , ion exchange chromatography
Ion exchangers are cation exchangers (eg carboxymethylcellulose; CM- cellulose) and anion exchangers (diethylaminoethylcellulose; DEAE?FONTCOLOR: black; FONT-SIZE: 10.5pt; ">Song "LANG="ZH-CN"> Cellulose), when the separated protein solution flows through the ion exchange chromatography column, the protein with the opposite charge to the ion exchanger is adsorbed on the ion exchanger, and then the pH is changed. Or the ionic strength method elutes the adsorbed protein.
(4) Separation methods based on different solubility of proteins
1 , salting out of protein
Neutral salt has a significant effect on the solubility of protein. Generally, at low salt concentration, the solubility of protein increases with the increase of salt concentration. This is called salt solution. When the salt concentration continues to increase, the solubility of protein decreases to varying degrees. Precipitation, this phenomenon is called salting out, adding a large amount of salt to the protein solution. High concentration of salt ions (such as ammonium sulfate SO4 and NH4 ) has a strong hydration power, which can capture the hydration layer of protein molecules. The " water loss " , so the protein colloids condense and precipitate. In the case of salting out, if the pH of the solution is at the isoelectric point of the protein, the effect is better. Since various protein molecules have different particle sizes and hydrophilic degrees, the salt concentration required for salting out is also different. Therefore, adjusting the neutral salt concentration in the mixed protein solution can precipitate various proteins in stages.
Factors affecting salting out are: ( 1 ) Temperature: In addition to temperature-sensitive proteins operating at low temperatures ( 4 degrees), they can generally be carried out at room temperature. Generally, the temperature is low and the protein solubility is lowered. However, some proteins (such as hemoglobin, myoglobin, albumin) have lower solubility at higher temperatures ( 25 degrees) than 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 (co-sinking phenomenon). Therefore, before the salting out, the serum should be diluted with the same amount of physiological saline to make the protein content 2.5-3.0% .
The neutral salts commonly used for protein salting out are mainly ammonium sulfate, magnesium sulfate, sodium sulfate, sodium chloride, sodium phosphate and the like. Wherein the most widely used ammonium sulfate, it has the advantage of high solubility and a small temperature coefficient (25 degrees saturated solution of 4.1M, i.e. 767 g / l; 0 to 3.9M saturation solubility, i.e. 676 g / liter), in In this solubility range, many proteins and enzymes can be salted out; in addition, the ammonium sulfate partial salting effect is 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 , it is adjusted with sulfuric acid or ammonia water.
After the protein is separated and separated by salting out, the salt in the protein needs to be removed. The common method is dialysis, that is, the protein solution is put into the cloning bag (usually cellophane), dialyzed with buffer, and continuously replaced. Buffers are preferably carried out at low temperatures because of the long time required for dialysis. In addition , the salt can be removed by the glucose gel G-25 or G-50 column, and the time used is relatively short.
2 , isoelectric precipitation method
When the protein is in an electrostatic state, the electrostatic repulsion between the particles is the smallest, so the solubility is also the smallest, and the isoelectric points of various proteins are different. The pH of the solution can be adjusted to reach the isoelectric point of a certain protein to precipitate, but this method is very It can be used alone in combination with salting out.
3 , low temperature organic solvent precipitation method
Using a water-miscible organic solvent, methanol, ethanol or acetone, the solubility of most proteins is reduced and precipitated. This method has higher resolution than salting out, but the protein is more susceptible to denaturation and should be carried out at low temperatures.
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