What are the common methods for protein isolation and identification?

Common protein identification methods are:

1. The easiest way is to burn the object to be identified.

2. Chemical reactions using chemical agents to identify proteins.

Third, the more accurate method is to use instruments for protein identification, such as mass spectrometers, which often encounter some problems about protein identification in biological research, such as the identification of single proteins or simple mixtures; sequence analysis of single proteins, etc. This type of protein identification requires high precision, so almost all of them use mass spectrometry instruments to accurately identify proteins.

Mass spectrometry is one of the platform techniques for the identification of proteins. Available mass spectrometers include Thermo Fisher's Q Exactive Mass Spectrometer, LTQ Orbitrap Velos Mass Spectrometer, and AB SciEx's 6500 Q Trap Mass Spectrometer. The use of hardware brands will be different depending on the appraisal agency.

The process of protein identification is generally divided into three steps: protein extraction, purification, and identification. This process is a process of "dissection" of proteins layer by layer, from which we can determine the molecular weight of proteins, identify protein glue dots, beads, IP sample proteins, non-variable mass spectrometry analysis and pull-down target protein spectrum identification, etc., and analyze various substances and properties in proteins in more detail.

1.The smell of burnt feathers produced by burning is protein.

2.When concentrated Hno3 is denatured, the yellow insoluble matter is a protein.

3.Refining and filtration, taking the filtrate, adding NaOH first, and then adding the biuret reagent, there is a purple reaction, which is a protein.

In general, the easiest way to identify proteins is in one of two cases:

1.Inspect solid substances: the easiest way is to burn them and smell them for the smell of burnt feathers, some contain protein, and none do not;

2.Test liquid substance: the easiest way is to add an appropriate amount of table salt or easily available soluble salt around you and mix it well to see if it is coagulated.

The main methods for separation according to the size of protein molecules include dialysis, ultrafiltration, centrifugation and gel filtration.

Dialysis and ultrafiltration are common methods for separating proteins.

Dialysis involves placing the mixture to be separated into a dialysis bag made of a semi-permeable membrane and then immersed in the dialysate for separation. Ultrafiltration is the process of using centrifugal force or pressure to force water and other small molecules through a semipermeable membrane, while proteins are trapped on the semipermeable membrane.

Both methods can separate protein macromolecules from small molecules, which are dominated by inorganic salts.

They are often used in combination with salting out and salting-out methods, both of which can be used to remove introduced inorganic salts after salting out or salting-out.

In the ultrafiltration process, the surface of the filter membrane is easily clogged by adsorbed proteins, so that the ultrafiltration speed slows down, and the molecular weight of the intercepted material becomes smaller and smaller. Therefore, when using the ultrafiltration method, it is necessary to choose the appropriate filter membrane, and tangential flow filtration can also be selected to obtain more ideal results.

1. Salting-out and organic solvent precipitation: adding a large amount of neutral salt to the protein solution to destroy the colloidal properties of the protein and precipitate the protein from the solution, which is called salting-out. Commonly used neutral salts are:

Ammonium sulfate, sodium chloride, sodium sulfate, etc. When salting out, the pH of the solution works best at the isoelectric point of the protein. Any organic solvent that can be mixed with water in any proportion, such as ethanol, methanol, acetone, etc., can cause protein precipitation.

2. Electrophoresis: Protein molecules have a net negative or positive charge in a solution above or below their pi, so they can move in the electric field. The magnitude of electrophoretic mobility depends mainly on the amount of charge carried by the protein molecule and the size of the molecule.

3. Dialysis method: The ultrafiltration properties of the dialysis bag membrane can be used to separate macromolecular substances from small molecules.

4. Chromatography: use the difference in the physical and chemical properties of each component in the mixture to separate the distribution between the two phases (stationary phase and mobile phase) in contact with each other. There are mainly ion exchange chromatography, gel chromatography, adsorption chromatography and affinity chromatography, among which gel chromatography can be used to determine the molecular weight of proteins.

5. Molecular sieve: also known as gel filtration method, the protein solution is added to the top of the column, let it leak downward, and the small molecule protein enters the pores, so it stays in the column for a long time, and the macromolecular proteins cannot enter the pores and radial out, so the proteins of different sizes can be separated.

6. Ultracentrifugation: Using the different density of substances, after ultracentrifugation, they are distributed in different liquid layers and separated. Ultracentrifugation can also be used to determine the molecular weight of a protein, which is proportional to its sedimentation coefficient s.