Salt chromatographic graded separation and gel chromatographic desalting of proteins
Salt chromatographic graded separation and gel chromatographic desalting of proteins
The purpose of this experiment is to understand the basic principles and operation of salting out graded separation of proteins; to understand the basic principles of dextran gel SephadexG-25 desalting and the preparation and elution techniques of gel columns as well as to understand the use of 752 UV-visible spectrophotometer.
Operation method
protein salting out
Principle
With a large number of neutral salts to make the protein precipitation process from the solution is called protein salting out. Proteins are hydrophilic colloids, under the influence of high concentrations of neutral salts to remove the hydration layer, at the same time, the protein molecules are neutralized by the charge, the result is that the colloidal stability of proteins was destroyed and precipitation precipitation. After dialysis or dilution with water can be soluble, so the salting out of proteins is a reversible process. The neutral salt concentration required for the salting out of different proteins is related to the type of protein and its pH. Large molecular weight proteins (e.g., globulins) are more easily precipitated than small molecular weight proteins (e.g., albumin). By varying the salt concentration, proteins of different molecular weights are precipitated separately. When the salt-containing protein solution flows through the gel chromatography column, the small molecular weight salt molecules move downward slowly because they enter into the micropores of the gel particles; while the large molecular weight proteins can not enter into the micropores of the gel particles, and they flow through the gel column at a faster speed, thus separating the proteins from the salt. Protein molecules have aromatic amino acid residues, therefore, the maximum absorption of 280 nm UV light, and proportional to the Beer's law. The protein samples were collected by UV spectrophotometer.
Materials and Instruments
Egg white Serum Move 1. Separation of ovalbumin Caveat 1. The amount of solid ammonium sulfate to be added in the above procedure can be calculated by referring to the appendix. 2. The height of the gel should be about 25 cm when loading the column, otherwise the desalination will not be good due to the small volume. 3. After measurement, do not pour out the sample, collect the sample with high protein content, seal it and store it in the refrigerator. 4. The gel column used once can be used again after equilibration. However, if the column has been used for several times, it should be regenerated (soak in low concentration of alkali for 30 min, rinse in water to neutral, soak in low concentration of acid for 30 min, rinse in water to neutral, load the column and use it, or store it in the refrigerator with preservative. For more product details, please visit Aladdin Scientific website.
Saturated Ammonium Sulfate Solution Ammonium Sulfate Dextran Gel SephadexG-25
Test tubes Triangular funnels Glass rods Filter paper Test tube holders 752 UV spectrophotometer Quartz cuvettes Glass chromatography columns Latex tubes Stoppers
(1)Take about 2 ml of ovalbumin in a test tube, add an equal volume of saturated ammonium sulfate solution, stir well, the protein precipitation, standing, filtered with filter paper to clarify the filtrate, the precipitate is ovoglobulin, the precipitate will be washed with 2 ml of half-saturated ammonium sulfate once.
(2) will precipitate out the filtrate after the ovalbumin into a test tube, and then add solid ammonium sulfate to make it saturated, observe whether there is no precipitation, if precipitation, then filter it. Filtered precipitate is ovalbumin.
2. Desalination (1) the preparation of the gel column: weigh the dextran gel sephadexG-25 5 g, add 80 ml of eluent (distilled water), dissolved in a boiling water bath for 30 min, with the method of pouring to remove the suspended particles. Then put into the inner diameter of 1.2 cm, height 30 cm glass. Then loaded into a glass chromatography column with an inner diameter of 1.2 cm and a height of 30 cm. Pay attention to filling uniformly, no bubbles and cracks exist, and keep the liquid level above the surface of the gel bed.
(2) Sample addition and elution: Open the outlet of the column and let the liquid flow out slowly until the liquid level is level with the surface of the gel bed. Then add 2 ml of salt-containing protein solution until the sample level reaches the surface of the gel bed, then gradually add 30 ml of eluent at 0.5~1 ml/min, and then add the sample at 0.5~1 ml/min. The elution was performed at a flow rate of 0.5~1 ml/min, and every 5 ml was collected in tubes step by step.
(3) The protein content of the collected solution can be detected by ultraviolet absorption at 280 nm. Salts can be identified by ionic coloration.
(4) Draw the absorption curve of protein at 280 nm, and collect the protein solution for use.
