Method for collecting concentrated protein in cell culture fluid
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Q: I want to use the supernatant to make Elisa , but the protein concentration in the supernatant is not enough. Is there any way to concentrate the protein ?
A: Protein concentration technology is a commonly used method in immunology. Several commonly used concentration techniques are introduced.
1 , dialysis bag concentration method
The use of a dialysis bag to concentrate the protein solution is one of the applications of Zui. Place the protein solution to be concentrated in a dialysis bag (no dialysis bag can be replaced with cellophane), ligation, and polymer ( 6 000 - 12 000 ) polymers such as polyethylene glycol (carbon wax), polyvinylpyrrole, alkanone, etc. Sucrose can be sprinkled outside the dialysis bag. The water absorbing agent can also be formulated into a solution of 30 % to 40 % concentration, and the dialysis bag containing the protein solution can be placed. After the water absorbing agent is used, it can be placed in a thermostat for drying or natural drying and can be reused.
2 , freeze-drying concentration method
This is a better way to concentrate proteins, which not only make the protein less susceptible to denaturation, but also maintain the ingredients inherent in the protein. It is directly sublimated in the frozen state to remove moisture. The specific method is to freeze the protein solution at a low temperature and then dispose it in a desiccator (a desiccant such as NaOH , CaCl 2 and silica gel is contained in the desiccator ). Closed, quickly evacuated, and maintained in an evacuated state. A dry powder containing the protein can be obtained in a few hours. The dried protein is easy to store and can be used in any concentration when applied. Freeze drying can also be carried out using a lyophilizer.
3 , blow dry concentration method
The protein solution was placed in a dialysis bag and placed under an electric fan. This method is simple, but the speed is slow, and the temperature can not be too high, so do not exceed 15 Â°C .
4 , ultrafiltration membrane concentration method
This method uses a microporous cellulose membrane to filter out water by high pressure, and the protein remains on the membrane for concentration purposes. There are two ways to concentrate: one is to insert the cellulose acetate membrane into the high pressure filter and filter under constant stirring; the other is to put the protein solution into the dialysis bag and built in the vent of the vacuum dryer, negative The air is pumped to make the liquid in the bag ooze out.
5 , gel concentration method
The gel with a smaller pore size, such as Sephadex G25 or G50 , is added directly to the protein solution. The amount of dry glue required is weighed according to the amount of water absorbed by the dry glue and the multiple of the protein solution to be concentrated. After being placed in a refrigerator, the gel particles are absorbed by water and then removed by centrifugation.
6 , concentrated gel concentration method
The concentrated gel is an organic polymer with a polymer network structure and has a strong water absorption property. Each gram of dry glue can absorb 120 ml-150 ml . It can absorb low molecular weight substances such as water, glucose, sucrose, inorganic salts, etc., and is suitable for concentrating biomacromolecules above 10,000 molecular weight. After concentration, the protein recovery rate can reach 80 % to 90 %. It is more convenient than concentrated glue and can be directly added to the concentrated solution. It must be noted that the pH of the concentrated solution should be greater than the isoelectric point of the concentrated material, otherwise cation exchange will occur on the surface of the concentrated gel, affecting the recovery rate of the concentrated material.
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