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1. Concentration of samples
During the preparation process, biological macromolecules become very dilute due to column purification. For storage and identification purposes, they often need to be concentrated. Commonly used concentration methods:
1. Vacuum concentration, evaporation and concentration
By lowering the liquid surface pressure, the boiling point of the liquid is reduced. The higher the vacuum degree of decompression, the lower the boiling point of the liquid and the faster the evaporation. This method is suitable for the concentration of some heat-resistant biological macromolecules.
2. Evaporation of air flow The flow of concentrated air can accelerate the evaporation of the liquid, spreading into a thin layer of solution, the surface continues to flow through the air; or the biological macromolecular solution is put into the dialysis bag and built into the cold room, and the electric fan is directed to blow the air The solvent passing through the membrane does not evaporate, but achieves the purpose of concentration. This method is slow in concentration and is not suitable for the concentration of a large number of solutions.
3. Frozen macromolecules form ice at low temperature. Salts and biomolecules do not enter the ice but remain in the liquid phase. During operation, the solution to be concentrated is cooled to make it solid, and then slowly melted. Using the difference between the melting point of the solvent and the solute to achieve the purpose of removing most of the solvent. For example, when the salt solution of protein and enzyme is concentrated by this method, pure ice crystals without protein and enzyme float on the liquid surface, and the protein and enzyme are concentrated in the lower solution, and the upper ice block is removed to obtain the concentration of protein and enzyme. liquid.
4. Absorption method The solution molecules in the solution are directly collected and concentrated by the absorbent. The absorbent used must not chemically react with the solution and does not adsorb biological macromolecules and is easily separated from the solution. Commonly used absorbents include polyethylene glycol, polyvinylpyrrolidone, sucrose, and gel. When using polyethylene glycol absorbents, first put the biomacromolecule solution in a semi-permeable membrane bag, and add polyethylene glycol The alcohol cover is placed at 4 degrees, the solvent in the bag oozes out and is quickly absorbed by the polyethylene glycol. After the polyethylene glycol is saturated with water, it must be replaced with a new one until the required volume is reached.
5. The ultrafiltration method uses a special membrane to selectively filter various solute molecules in the solution. When no liquid passes through the membrane under a certain pressure (nitrogen pressure or vacuum pump pressure), the solvent and small molecules penetrate, Macromolecules are blocked and retained. This is a new method developed in recent years. It is most suitable for the concentration or desalination of biological macromolecules, especially proteins and enzymes. It has low cost, convenient operation, mild conditions, and can better maintain biological macromolecules. The activity, high recovery rate and other advantages. The key to the application of ultrafiltration is the choice of membranes. Different types and specifications of membranes, water flow rate, molecular weight cut-off value (that is, the minimum molecular weight value of molecules that can be retained by the membrane) and other parameters are different, and must be selected according to the needs of the work. In addition, the form of the ultrafiltration device, solute composition and properties, solution concentration, etc. all have a certain influence on the ultrafiltration effect. Diaflo ultrafiltration membrane molecular weight cutoff:
Membrane name Molecular weight cutoff value Large average diameter of pores
XM-300 300,000 140
XM-200 100,00055
XM-50 50,000 30
PM-30 30,000 22
UM-20 20,00018
PM-10 10,000 15
UM-2 1,000 12
UM05 500 10
A hollow fiber tube is made with the above ultrafiltration membrane, and many such tubes are gathered into a bundle, and both ends of the tube are connected with a buffer solution of low ionic strength, so that the buffer solution continuously flows in the tube. The fiber tube is then immersed in the protein solution to be dialyzed. When the buffer flows through the fiber tube, small molecules can easily diffuse through the membrane and large molecules cannot. This is the fibrous filtration method, because the dialysis area is increased, so the dialysis time is shortened by 10 times.
Second, dry
The products prepared by biological macromolecules, in order to prevent deterioration, easy to store, often require drying treatment, the most commonly used methods are freeze drying and vacuum drying. Vacuum drying is suitable for drying and storage of substances that are not resistant to high temperature and easy to oxidize. The whole device includes a dryer, condenser and vacuum drying principle, while increasing the temperature factor. At the same pressure, the water vapor pressure drops as the temperature drops, so at low temperatures and pressures, ice easily sublimates into gas. During operation, the liquid to be dried is generally frozen below the freezing point to make it solid, and then the solvent is turned into gas at low temperature and low pressure to be removed. The dried product of this method has the advantages of looseness, good solubility, and maintaining the natural structure. It is suitable for the dry storage of various biological macromolecules.
3. Storage
The stability of biological macromolecules has a great relationship with the preservation method. Dried products are generally stable, and their activity can not change significantly in days or even years at low temperatures. The storage requirements are simple, as long as the dried samples are placed in a desiccator (with a desiccant inside) to seal and keep 0-4 It can be stored in a refrigerator. Pay attention to the following points when storing in liquid state.
1. The sample should not be too dilute. It must be concentrated to a certain concentration before packaging and storage. If the sample is too dilute, it will easily denature the biological macromolecules.
2. Generally, preservatives and stabilizers need to be added. Commonly used preservatives include toluene, benzoic acid, chloroform, and thymol. Common stabilizers for proteins and enzymes include ammonium sulfate paste, sucrose, glycerin, etc., such as enzymes, substrates and coenzymes can also be added to improve their stability. In addition, calcium, zinc, boric acid and other solutions also have certain protective effects on certain enzymes. Nucleic acid macromolecules are generally stored in standard buffers of sodium chloride or sodium citrate.
3. The storage temperature requirements are low, most of them are stored in the refrigerator at about 0 degrees, and some are lower, depending on different materials.
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