The main factors affecting electrophoretic separation:
1. Nature of biological macromolecules to be separated: The charge, molecular size and nature of the biomacromolecules to be separated will have a significant effect on electrophoresis. In general,
The larger the charge of the subband, the smaller the diameter, and the more spherical the shape, the faster the electrophoretic migration.
2. The nature of the buffer: The pH of the buffer will affect the degree of dissociation of the biological macromolecules to be separated, thereby affecting its charge properties. The farther the pH value of the solution is from its isoelectric point, the amount of net charge it will carry. The greater the electrophoresis speed, the greater the rate of electrophoresis, especially for proteins such as amphipathic molecules, the buffer pH will also affect the direction of its electrophoresis, when the buffer pH is greater than the isoelectric point of protein molecules, protein molecules are negatively charged, its electrophoretic The direction is pointing to the positive pole. In order to maintain the charge of the biological macromolecules to be separated during electrophoresis and the stability of the buffer pH, the buffer usually has to maintain a certain ionic strength, generally 0.02-0.2. If the ionic strength is too low, the buffering capacity is poor, but if the ion If the intensity is too high, a strong oppositely charged ion diffusion layer (ie, ionic atmosphere) will form around the molecules to be separated. Since the ionic atmosphere and the molecules to be separated move in opposite directions, electrostatic attraction occurs between them, thus causing electrophoresis. The speed is reduced. In addition, the viscosity of the buffer also affects the electrophoretic speed.
3. Electric field strength : Electric field strength (V/cm) is the potential drop per centimeter, also called potential gradient. The greater the electric field strength, the faster the electrophoresis. However, increasing the electric field strength will cause the current intensity through the medium to increase, resulting in increased heat generation during the electrophoresis process. The work (W) of the current in the medium is: W=I2.Rt where: I is the current intensity, R is the resistance, and t is the electrophoresis time.
Most of the work done by the current is converted to heat, which causes the temperature of the medium to rise, which has many implications:
1. The diffusion rate of the sample and buffer ions increases, causing widening of the sample separation band;
2, produce convection, causing the mixture of the objects to be separated;
3, if the sample is sensitive to heat, it will cause protein denaturation;
4, cause the medium to reduce the viscosity, resistance drop and so on. The heat generated in the electrophoresis is usually emitted from the center to the periphery, so the center temperature of the medium is generally higher than the outer circumference, especially in the tube electrophoresis. This causes the viscosity of the central part of the medium to decrease relative to the outer part, the friction coefficient decreases, and the electrophoretic migration speed. Increasing, the electrophoretic separation band is generally bow-shaped because the electrophoretic velocity of the central part is faster than the edge. Decreasing the current intensity can reduce heat generation, but it will prolong the electrophoresis time and cause the diffusion of the biological macromolecules to be separated to affect the separation effect. Therefore, appropriate electric field strength should be selected in the electrophoresis experiment, and at the same time, the temperature can be appropriately reduced to obtain a better separation effect.
4. Electroosmosis: The relative movement of a liquid in an electric field to a solid support medium is called electroosmosis. Because there may be some charged groups on the surface of the support medium, such as the filter paper surface usually has some carboxyl groups, the agar may contain some sulfate groups, and the glass surface usually has Si-OH groups and so on. After ionizing these groups, the surface of the supporting medium will be charged, and some ions with opposite charges will be adsorbed. Under the action of the electric field, the ions will move toward the electrode to form the flow of solution on the surface of the medium. This phenomenon is electroosmosis. When the pH value is higher than 3, the surface of the glass is negatively charged and the positive ions in the solution are adsorbed, causing the solution layer in the vicinity of the glass surface to become positively charged. Under the action of the electric field, it migrates to the negative electrode to drive the electroosmotic flow of the electrode solution to the negative electrode. . If the electroosmotic direction is the same as the electrophoresis direction of the molecules to be separated, the electrophoresis speed is increased; if the electrophoretic direction is reversed, the electrophoresis speed is decreased.
5. Sieve support media: The size of the support media has a significant effect on the electrophoretic migration velocity of the biomolecules to be isolated. In the medium with large mesh, the swimming speed is fast. On the contrary, the swimming speed is slow.
A note on plastic recycling gel :
1. The electrophoresis buffers and gels are all new systems. The plastic cutting table is cleaned and the blades are preferably washed and sterilized. The general rule is to ensure that the cut tape has no foreign DNA contamination.
2. The glue is to recover all the glue at the location of the entire destination. In order to reduce the volume of glue, you can use a relatively thin glue to do as long as you can sample enough, you can also use thin and wide comb to run glue.
3. About protection, it is enough after general plexiglass, wear a protective mask to protect the eyes, if you wear resin glasses on it. If you are very afraid of ultraviolet radiation, or have special requirements for the glue, such as the requirement that there is no EB, you can use the dot marker and the scale with a ruler to determine the position of the destination tape on the glue to cut the glue.
4. Follow the normal program marker to run the glue, then cut off the glue with the marker, stain the EB, and take a photo with the ruler with a scale under the UV lamp. Since the relative position between the tape to be recovered and the marker is known, the position of the target tape on the unstained rubber can be measured according to the ruler. If you find it difficult to judge, you can spot a small amount directly on the edge of the marker so that the position is easily determined.
1. Nature of biological macromolecules to be separated: The charge, molecular size and nature of the biomacromolecules to be separated will have a significant effect on electrophoresis. In general,
The larger the charge of the subband, the smaller the diameter, and the more spherical the shape, the faster the electrophoretic migration.
2. The nature of the buffer: The pH of the buffer will affect the degree of dissociation of the biological macromolecules to be separated, thereby affecting its charge properties. The farther the pH value of the solution is from its isoelectric point, the amount of net charge it will carry. The greater the electrophoresis speed, the greater the rate of electrophoresis, especially for proteins such as amphipathic molecules, the buffer pH will also affect the direction of its electrophoresis, when the buffer pH is greater than the isoelectric point of protein molecules, protein molecules are negatively charged, its electrophoretic The direction is pointing to the positive pole. In order to maintain the charge of the biological macromolecules to be separated during electrophoresis and the stability of the buffer pH, the buffer usually has to maintain a certain ionic strength, generally 0.02-0.2. If the ionic strength is too low, the buffering capacity is poor, but if the ion If the intensity is too high, a strong oppositely charged ion diffusion layer (ie, ionic atmosphere) will form around the molecules to be separated. Since the ionic atmosphere and the molecules to be separated move in opposite directions, electrostatic attraction occurs between them, thus causing electrophoresis. The speed is reduced. In addition, the viscosity of the buffer also affects the electrophoretic speed.
3. Electric field strength : Electric field strength (V/cm) is the potential drop per centimeter, also called potential gradient. The greater the electric field strength, the faster the electrophoresis. However, increasing the electric field strength will cause the current intensity through the medium to increase, resulting in increased heat generation during the electrophoresis process. The work (W) of the current in the medium is: W=I2.Rt where: I is the current intensity, R is the resistance, and t is the electrophoresis time.
Most of the work done by the current is converted to heat, which causes the temperature of the medium to rise, which has many implications:
1. The diffusion rate of the sample and buffer ions increases, causing widening of the sample separation band;
2, produce convection, causing the mixture of the objects to be separated;
3, if the sample is sensitive to heat, it will cause protein denaturation;
4, cause the medium to reduce the viscosity, resistance drop and so on. The heat generated in the electrophoresis is usually emitted from the center to the periphery, so the center temperature of the medium is generally higher than the outer circumference, especially in the tube electrophoresis. This causes the viscosity of the central part of the medium to decrease relative to the outer part, the friction coefficient decreases, and the electrophoretic migration speed. Increasing, the electrophoretic separation band is generally bow-shaped because the electrophoretic velocity of the central part is faster than the edge. Decreasing the current intensity can reduce heat generation, but it will prolong the electrophoresis time and cause the diffusion of the biological macromolecules to be separated to affect the separation effect. Therefore, appropriate electric field strength should be selected in the electrophoresis experiment, and at the same time, the temperature can be appropriately reduced to obtain a better separation effect.
4. Electroosmosis: The relative movement of a liquid in an electric field to a solid support medium is called electroosmosis. Because there may be some charged groups on the surface of the support medium, such as the filter paper surface usually has some carboxyl groups, the agar may contain some sulfate groups, and the glass surface usually has Si-OH groups and so on. After ionizing these groups, the surface of the supporting medium will be charged, and some ions with opposite charges will be adsorbed. Under the action of the electric field, the ions will move toward the electrode to form the flow of solution on the surface of the medium. This phenomenon is electroosmosis. When the pH value is higher than 3, the surface of the glass is negatively charged and the positive ions in the solution are adsorbed, causing the solution layer in the vicinity of the glass surface to become positively charged. Under the action of the electric field, it migrates to the negative electrode to drive the electroosmotic flow of the electrode solution to the negative electrode. . If the electroosmotic direction is the same as the electrophoresis direction of the molecules to be separated, the electrophoresis speed is increased; if the electrophoretic direction is reversed, the electrophoresis speed is decreased.
5. Sieve support media: The size of the support media has a significant effect on the electrophoretic migration velocity of the biomolecules to be isolated. In the medium with large mesh, the swimming speed is fast. On the contrary, the swimming speed is slow.
A note on plastic recycling gel :
1. The electrophoresis buffers and gels are all new systems. The plastic cutting table is cleaned and the blades are preferably washed and sterilized. The general rule is to ensure that the cut tape has no foreign DNA contamination.
2. The glue is to recover all the glue at the location of the entire destination. In order to reduce the volume of glue, you can use a relatively thin glue to do as long as you can sample enough, you can also use thin and wide comb to run glue.
3. About protection, it is enough after general plexiglass, wear a protective mask to protect the eyes, if you wear resin glasses on it. If you are very afraid of ultraviolet radiation, or have special requirements for the glue, such as the requirement that there is no EB, you can use the dot marker and the scale with a ruler to determine the position of the destination tape on the glue to cut the glue.
4. Follow the normal program marker to run the glue, then cut off the glue with the marker, stain the EB, and take a photo with the ruler with a scale under the UV lamp. Since the relative position between the tape to be recovered and the marker is known, the position of the target tape on the unstained rubber can be measured according to the ruler. If you find it difficult to judge, you can spot a small amount directly on the edge of the marker so that the position is easily determined.
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