How to make good use of protein purification buffer

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A good protein purification master can definitely become a soup master, and electrophoresis can only be done well if the soup is done well. The principle is almost similar. Regardless of whether you are cooking soup or purifying protein, you must fully understand the heat, the heat resistance of the protein, and the characteristics of the buffer.

Generally speaking, the heat resistance of a protein has little to do with the stability of its primary structure (the stability of the peptide bond). It is more that the protein aggregates and precipitates when the hydrophobic part of the higher structure is destroyed at high temperature and exposed. For example, an egg, even if it is made into frangipani soup, if it is dissolved in SDS loading buffer, most of the protein will still retain the stability of the primary structure.

However, the above statement is not absolute. For example, when boiling soup, as long as the time is in place, the soup can become delicious. This is actually the hydrolysis of protein polypeptide chains, releasing amino acids and some small molecules to make the taste delicious. In other words, as long as the soup or heating time is sufficient, the peptide chain of the protein will eventually be broken, and the higher the temperature, the faster the degradation.

Protein buffer composition and function

In order to improve the stability of the protein, many ingredients are added to the protein buffer, such as Tris-HCl (Trishydroxymethylaminomethane hydrochloride) or Tris glycine, and EDTA salt (Ethylenediaminetetraacetic acid disodium), and SDS is also required. , DTT (Dithiothreitol) and so on. As a reducing agent, DTT can protect the peptide bonds of proteins. Generally, peptide bonds are more stable than disulfide bonds. Without reducing agents, boiling samples at 50 degrees, 80 degrees, and 100 degrees for 5 minutes can cause some of the disulfide bonds to open. The higher the opening, the more. It can be seen that in the non-reducing loading buffer at 100 degrees, the disulfide bond opens slowly, and the peptide bond breaks more slowly.

If the reducing agent added in the buffer is too poor, re-crosslinking between the reducing agent and the protein will occur, that is, the oligomerized reducing agent and the sulfhydryl group on the protein will be cross-linked again. At this time, the cross-linking is non-specific Yes, the band will become slushy. Therefore, TCEP is used as a reducing agent in enzymes or demanding protein buffers.

When Tris glycine is used as the buffer, some laboratories will reuse the glycine running buffer, but it is not necessary. Generally, the quality of glycine used in electrophoresis is not good enough, and there are too many impurities. There is a problem when it is compressed in the upper end of the electrophoresis gel. When entering the separation gel, the ion pair will be imbalanced and the diffusion will be severe.

In many laboratories, SDS electrophoresis samples are boiled at 100 degrees for 3-5 minutes. Heating has little effect on protein peptide bonds, but the pH of Tris is affected by temperature. Desheng is a manufacturer of biological buffers and can provide high-quality Tris, MOPS and other buffer raw materials.