Some notes of Electrophoresis experiment
Main factors affecting electrophoretic separation:
1. Properties of biomacromolecules to be separated: the charge, molecular size and properties of biomacromolecules to be separated will have a significant impact on electrophoresis. Generally speaking, the larger the charge, the smaller the diameter and the closer the spherical shape of the subband, the faster the electrophoretic migration rate.
2. Properties of buffer: Buffer pH can affect the degree of the separation of biological macromolecules, which affect the electric properties, solution pH value from its isoelectric point away, the greater the amount of the net charge they carry, electrophoresis rate is, the greater the especially for proteins such as amphoteric molecules, buffer pH also will affect the direction of electrophoresis, when buffer pH is greater than the isoelectric point of protein molecules, Protein molecules are negatively charged and their electrophoretic direction is positive. In order to keep the electrophoresis process for separation of biological macromolecules charge and the stability of the buffer pH value, buffer to keep constant ionic strength, usually in 0.02 to 0.2, ionic strength is too low, the buffering capacity is poor, but if the ionic strength, would stay in the separation of molecules formed around the stronger with the opposite charge of ion diffusion layer (i.e., ion atmosphere), Because the ionic atmosphere and the molecules to be separated move in the opposite direction, the electrostatic attraction between them causes the electrophoretic speed to decrease. In addition, the viscosity of the buffer also affects the electrophoretic speed.
3. Electric field intensity: Electric field intensity (V/cm) is the potential drop per centimeter, also known as potential gradient. The higher the electric field intensity, the faster the electrophoresis speed. However, increasing the intensity of the electric field will increase the intensity of the current passing through the medium, resu
4. The sample and buffer ion diffusion rate increases, resulting in the sample separation band widened;
5. Generate convection, cause the mixture of the separation;
6. If the sample is sensitive to heat, it will cause protein denaturation;
7. Cause medium viscosity reduction, resistance drop and so on. Heat generated in electrophoresis is usually send out from the center to the peripheral, so the media center to the peripheral temperature, especially for tubular electrophoresis, the resulting relative to the central part of the medium viscosity decreased, the week outside friction coefficient decreases, and electrophoretic migration velocity increases, due to the central part of the electrophoresis faster than the edge, so the electrophoresis separation zone usually bow type. Reducing the current intensity can reduce the heat generation, but it will prolong the electrophoresis time, cause the increase of the diffusion of biological macromolecules to be separated and affect the separation effect. Therefore, appropriate electric field intensity should be selected in the electrophoresis experiment, and the temperature can be lowered appropriately to obtain a better separation effect.
8. Electroosmosis: liquid in the electric field, for the solid supporting medium relative movement, known as electroosmosis phenomenon. There may be some charged groups on the surface of the supporting medium, such as some carboxyl groups on the surface of filter paper, some sulfuric acid groups in AGAR, and Si-OH groups on the surface of glass. After ionizing, these groups will make the surface of the supporting medium charged, adsorb some ions with opposite charges, move to the electric direction under the action of the electric field, and form the flow of the solution on the surface of the medium. This phenomenon is electroosmosis. When the pH value is 3, the glass surface is negatively charged and absorbs positive ions in the solution, causing the solution layer near the glass surface to be positively charged.
