Protein electrophoresis (SPE) is a protein analysis technology. Proteins with negative or positive charge in buffer and moving to anode in electric field are called electrophoresis. Different protein molecules have different electrophoretic mobility. Common protein electrophoresis was paper electrophoresis, starch gel electrophoresis, agarose gel electrophoresis, cellulose acetate gel electrophoresis, and polyacrylamide gel electrophoresis.
Among them, sodium dodecyl sulfate polyacrylamide gel electrophoresis (referred to as SDS-PAGE) is the most commonly used protein expression analysis technology in polyacrylamide gel electrophoresis (sulfate). The principle of this technology is to separate the protein in the electrophoresis gel according to the different molecular weight of the protein in the sample.
Proteins contain a lot of amino (+) and carboxyl (-), and different proteins show different charges at different pH values. In order to make the mobility of proteins in electrophoresis only related to molecular weight, we usually carry out some treatment (loading buffer) before loading. That is, adding SDS and β- Upper buffer of mercaptoethanol.
SDS is sodium dodecyl sulfonate （CH3-(CH2)10-CH2OSO3– Na+, which is an anionic surfactant. It can break the hydrogen bonds within and between molecules and destroy the secondary and tertiary structures of protein molecules; β- Mercaptoethanol is a strong reducing agent, which can break the disulfide bond between cysteine residues.
After the electrophoretic sample is added into the sample treatment solution and treated at high temperature, its purpose is to fully combine SDS with protein, so as to completely denature and depolymerize the protein, form a rod structure, and negatively charge the whole protein at the same time; In addition, bromophenol blue dye is usually added to the sample treatment solution to monitor the whole electrophoresis process; In addition, an appropriate amount of sucrose or glycerol is added to the sample treatment solution to increase the solution density, so that the sample solution can quickly sink into the bottom of the sample groove when adding samples. When the sample is put on and connected to the current between the two poles (the anode above the electrophoresis cell is negative, the cathode is below), the mobile interface is formed in the gel, and the polypeptide complex containing SDS negatively charged in the gel is propelling to the positive electrode. Firstly, the SDS polypeptide complex contained in the sample is aggregated into a very thin zone (or accumulation layer) on the surface of the separation gel through the highly porous concentrated gel. When the protein enters the separation gel, other ions are at pH 8 In the solution of 8, it quickly reaches the positive electrode, and only protein molecules move slowly in the separation gel. In the process of electrophoresis, the pH value of protein changes due to the change of solution ions, but at each moment, the number of charges divided by the unit mass is different. Therefore, those with more negative charges migrate faster, and vice versa, which shows the charge effect. Due to the small pore size of the gel and its integral sieve like structure, they have large resistance to macromolecules and small resistance to small molecules, playing a molecular sieve effect, that is, proteins in the separation gel have differences in mobility due to molecular sieve effect and charge effect, and finally separate from each other.
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