Electrophoresis refers to the charged test sample (protein, nucleotide, etc.) in an inert support medium (such as paper, cellulose acetate, agarose gel, polyacrylamide gel, etc.) under the action of an electric field, the method of moving to the corresponding electrode direction at respective speeds, separating the components into narrow zones, recording the electrophoresis zone map or calculating the percentage thereof by a suitable detection method.

The basic principles and classification of electrophoresis technology:

In an electric field, the force (F) that pushes the charged particle motion is equal to the product of the net charge (Q) and the electric field strength (E) of the particle. The forward movement of F=QE particle is also affected by the resistance (F). For a spherical particle, it obeys Stoke's law, ie: F′=6πrην where r is the particle radius, η is the medium viscosity, and ν is the particle moving speed. When the particle moves stably in the electric field: F=F' is QE=6πrην

It can be seen that the mobility of a spherical particle depends first on its own state, that is, proportional to the amount of electricity it is charged, and inversely proportional to its radius and viscosity of the medium. In addition to their own state factors, other factors in the electrophoretic system also affect the electrophoretic mobility of the particles.

Electrophoresis can be divided into two categories: free electrophoresis (no support) and zone electrophoresis (with support). The former includes Tise-leas microelectrophoresis, microelectrophoresis, isoelectric focusing electrophoresis, isotachophoresis and density gradient electrophoresis. Zone electrophoresis includes filter paper electrophoresis (normal pressure and high pressure), thin layer electrophoresis (film and sheet), gel electrophoresis (agar, agarose, starch gel, polyacrylamide gel).

The development of free electrophoresis is not rapid because of its complex structure, large size, strict operation requirements, and high price. The zone electrophoresis can be used as a support for various types of substances, and its application is extensive. This section describes only a few of the commonly used zone electrophoresis.

Factors affecting electrophoretic mobility:

1. Electric field strength Electric field strength refers to the potential drop per unit length (cm), also called the potential gradient. If the filter paper is used as a support, both ends are immersed in the electrode liquid, the paper length at the interface between the electrode liquid and the filter paper is 20 cm, and the measured potential drop is 200 V, then the electric field strength is 200 V / 20 cm = 10 V / cm. When the voltage is below 500V and the electric field strength is 2-10v/cm, it is normal pressure electrophoresis. The voltage is above 500V, and the electric field strength is 20-200V/cm for high-pressure electrophoresis. The electric field strength is large, and the mobility of charged particles is accelerated, so it is time-saving, but due to the generation of a large amount of heat, a cooling device should be provided to maintain the constant temperature.

2. pH of the solution The pH of the solution determines the degree of dissociation of the separated material and the charged nature of the particle and the amount of net charge carried. For example, a protein molecule, which is an ampholyte having both an acidic group (-COOH) and a basic group (-NH2), has a positive and negative charge in a solution, that is, the net charge of the molecule is equal to zero. At this time, the protein no longer moves in the electric field, and this pH of the solution is the isoelctric point (pI) of the protein. If the pH of the solution is on the acid side of the isoelectric point, ie pH < pl, the protein is positively charged and moves to the negative electrode in the electric field. If the pH of the solution is at the isoelectric side of the isoelectric point, ie, pH > pl, the protein is negatively charged and moves toward the positive electrode. The farther the pH of the solution is from pl, the more net charge the particle carries, and the greater the electrophoretic mobility. Therefore, when electrophoresis, the appropriate pH buffer should be selected according to the nature of the sample.

3. Ionic strength of the solution When the ion concentration in the electrophoresis liquid increases, the particle mobility decreases. The reason is that the charged particles attract the opposite ions to gather around them, forming an ionic atmosphere that is opposite to the moving particle. The ionic atmosphere not only reduces the charge of the particle, but also increases the resistance of the particle advancement, even making it impossible. Swimming. However, if the ion concentration is too low, the total concentration of the buffer and the buffer capacity will be lowered, the pH of the solution will not be maintained, the charge of the particle will be affected, and the migration speed will be changed. This barrier effect of ions is related to their concentration and valence. It can be expressed by the ionic strength I.

4. Electroosmosis The relative movement of a liquid to a solid support under the action of an electric field is called electro-osmosis. The reason for this is that the solid support is porous and has a dissociable chemical group, so the positive or negative ions in the solution are often adsorbed to make the solution relatively negatively or positively charged. When using filter paper as a support, the cellulose on the paper adsorbs OH- with a negative charge, and the aqueous solution in contact with the paper generates H3O+, and positively charges to the negative electrode. If the particle originally moves to the negative electrode in the electric field, the performance speed of the particle is obtained. It is faster than its inherent speed. If the mass point is originally moved to the positive electrode, the performance speed is slower than its inherent speed. It should be noted that the support with low electroosmosis should be selected as much as possible to reduce the influence of electroosmosis.

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