The process of precipitation from the solution by destroying the colloidal stability of the protein and reducing the solubility of the protein
Salting outIt refers to adding high concentration neutral salt into protein solution to destroy proteinColloidal stabilityThe process of precipitating protein from solution by reducing its solubility.SaliferousconcentrationWith the pH value of the solution, the proteins in the mixed solution can be salted out one by one. This operation of separating proteins is called subsection salting out.[1]For example: serum globulin (50% (NHfour)twoSOfourSaturation), albumin (saturation (NHfour)twoSOfour）。
Chinese name
fractional salting out
Purpose
Gradually separate the proteins in the mixture by salting out
For the salting out of the separated target protein, it is better to use subsection salting out.Because the solubility and isoelectric point of different proteins are different, the pH value and ionic strength required for precipitation are also different. By changing the concentration of salt and the pH value of solution, the proteins in the mixed solution can be salted out in batches. This method of separating proteins is called fractional salting out.For example, semi saturated ammonium sulfate can precipitate plasma globulin, and saturated ammonium sulfate can precipitate all proteins including plasma albumin.
principle
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Proteins are hydrocolloids, and the stability of the colloid is maintained by the hydration film and the isotropic charge (generally, proteins are negatively charged in solutions with pH 7.0).Add some alkali metal oralkaline-earth metalAnd neutral salts of, then charge neutralization (loss of charge) occurs.When the concentration of salt is high enough, the protein colloidal particles dehydrate and precipitate, which is called salting out.The precipitate obtained from salting out can be dissolved again and its original molecular structure can be restored after dialysis or dilution with water to reduce or remove saltbiological activityTherefore, the precipitation generated by salting out is reversible.The size and hydrophilicity of various protein molecules are different, so the salt concentration required for salting out is also different.By adjusting the salt concentration in the mixed protein solution, various proteins can be precipitated in sections.Globulin can be precipitated in saturated ammonium sulfate solution, and albumin can be precipitated in saturated ammonium sulfate solution. This method isProtein separation and purificationCommon methods in the process.
The protein molecule is very large, and its particles are within the range of colloidal particles (1~100nm in diameter), so it cannot penetrateSemipermeable membrane。Select appropriate apertureSemipermeable membraneBecause small molecular substances can penetrate, but protein particles cannot, it can separate proteins from small molecular substances.This method can remove neutral salts and other small molecular substances mixed with protein.This technology is calleddialysis, is commonly used to purify proteins.Salt outProtein precipitationThe original structure andbiological activity。[2]
Operation steps
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1. The conditions for staged salting out need to be explored by small experiments first, such as 0% - 30% ammonium sulfate precipitation, 30% - 60% ammonium sulfate precipitation, and finally 60% - 80% ammonium sulfate precipitation.
2. Each stage of salting out will be centrifuged, precipitated, dialyzed and tested for activity after standing.It can be seen from the experimental results that which section of salting out has the most target products and relatively fewer impurities.
3. For example, the active substances in the experiment are mainly produced in the 60% - 80% section. In future experiments, 0% - 60% ammonium sulfate precipitation can be carried out first, and this section of precipitate can be discarded (removing most impurities);Then, 60% - 80% ammonium sulfate precipitation is carried out. The precipitated substance is the substance with high content of the desired target substance, which is collected for the next step of purification.
