Protein purification and precipitation

Protein purification and precipitation

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Gel electrophoresis

The net charge of a protein, the molecular mass and the conformation determine the migration speed of this protein in an electric field. This is used in gel electrophoresis, in which the free movement of the charged proteins through the gel matrix is ​​hindered. Estimate the molecular mass of a protein or determine the purity of a protein preparation.

Gels made from the crosslinked polymer polyacrylamide are mainly used for the electrophoresis of proteins. This polymer acts like a kind of molecular sieve: in an electric field, the proteins are slowed down in their migration through the gel in roughly proportion to their molecular mass. If a protein is only to be separated according to its size and not according to its charge, the detergent sodium dodecyl sulfate (sodium dodecyl sulfate, SDS) added. SDS binds most proteins in roughly proportion to their molecular mass. The bound SDS thus causes a high negative net charge regardless of the intrinsic charge of the protein. In addition, the native conformation of the protein is changed by this detergent, so that the proteins all have a more or less similar conformation in the gel. SDS polyacrylamide gel electrophoresis (SDS polyacrylamide gel electrophoresis, SDS-PAGE) only separates proteins according to their molecular mass.

In the case of native polyacrylamide gel electrophoresis, no SDS is added, so the protein is separated in the gel according to molecular mass, conformation and charge. Although proteins often cannot be compared directly, this method has the advantage that the activity of a protein is usually retained.

After the electrophoresis is complete, the proteins are stained to make them visible.

Video: Protein Precipitation - Types, Methods, Principle, Advantages and Disadvantages (August 2022).