What are the methods for separating and purifying proteins

In daily life, we don't know much about some structural components and molecular weight components of the body. Because of this lack of understanding, people don't recognize them, which leads to some disagreements and affects people's bodies. In fact, the method of separating and purifying protein can be judged accordingly by understanding the separation method and the specific combination of components. Only by understanding this aspect can we know what it is composed of.

Separation methods

Dialysis is a method that uses a dialysis bag to separate large molecular proteins from small molecular compounds.

Ultrafiltration

Positive pressure or centrifugal force is used to make the protein solution pass through an ultrafiltration membrane with a certain molecular weight cutoff to achieve the purpose of concentrating the protein solution.

The precipitation method using organic solvents such as acetone and ethanol can destroy the hydration layer of protein and precipitate the protein at a low temperature of 0~4℃. Under the influence of adverse factors such as high ambient temperature, organic solvents can cause protein denaturation.

Salt precipitation is the process of adding ammonium sulfate, sodium sulfate or sodium chloride to a protein solution to neutralize the surface charge of the protein and destroy the hydration membrane, leading to protein precipitation.

Immunoprecipitation method: Utilizes the property of specific antibodies to recognize corresponding antigenic proteins and form antigen-antibody complexes to separate antigenic proteins from protein mixed solutions.

Electrophoresis: Proteins are charged particles in solutions above or below their pI and can move toward the positive or negative electrode in an electric field. The technique of separating proteins by allowing them to migrate in an electric field is called electrophoresis. Several important protein electrophoresis:

Electrophoresis operation

SDS-polyacrylamide gel electrophoresis is often used to determine the molecular weight of proteins.

Isoelectric focusing is an electrophoretic method that separates proteins based on differences in their isoelectric points.

Two-dimensional gel electrophoresis is an important technique in proteomics research.

Chromatography: When the protein solution to be separated (mobile phase) passes through a solid substance (stationary phase), the protein components are repeatedly distributed in the two phases according to the particle size, charge and affinity of the protein to be separated, and flow through the stationary phase at different speeds to separate the proteins. Gel filtration (molecular sieve, gel filtration; exclusion chromatography): separate proteins based on their molecular size.

Ion exchange: The charge and properties of proteins are different.

Cation exchanger: CM-cellulose

Anion exchanger: DEAE-cellulose

Affinity chromatography: antigen-antibody, ligand-receptor, metal ions, biotin, etc.

High performance liquid chromatography (HPLC): reverse phase HPLC, ion HPLC, gel filtration HPLC

Ultracentrifugation: Separation of proteins based on their molecular weight and shape.