Main factors affecting electrophoretic separation in laboratory standard test - Database & Sql Blog Articles

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Main factors affecting electrophoresis separation in laboratory standard test

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[Biological reagents:

Ethyl violet 2390-59-2

]

The nature of the biomacromolecule to be separated: the charge, molecular size and properties of the biomolecule to be separated will have a significant effect on electrophoresis. In general, the larger the charge amount of the molecular band, the smaller the diameter, and the closer the shape is to the spherical shape, the faster the electrophoretic migration speed.

2

[Biological reagents:

Ethyl violet 2390-59-2

Standard electrophoresis

The nature of the buffer used in the experiment:

The PH value of the buffer used in the experiment will affect the dissociation degree of the large water molecules to be separated, and thus the influence on the charging property. The farther the solution PH is from the isoelectric point, the larger the electrostatic charge is. The speed is also greater, especially for amphiphilic molecules such as proteins, the pH of the buffer also affects the direction of electrophoresis. When the pH of the buffer is greater than the isoelectric point of the protein molecule, the protein molecule is negatively charged, and the direction of electrophoresis is directed to the positive electrode. . In order to maintain the charge of the biomolecule to be separated during the electrophoresis and the stability of the pH of the buffer, the buffer usually maintains a certain ionic strength, generally at 0.02-0.2, and the ionic strength is too low, so the buffering capacity is poor.

3

[Biological reagents:

Ethyl violet 2390-59-2

Standard product during the experiment

The effect of electric field strength:

The electric field strength is the potential drop per centimeter, also called the potential gradient. The higher the electric field strength, the faster the electrophoresis speed. However, how large the electric field strength will increase the heat generated by the electrophoresis process due to the positive current intensity of the ring.

The total current made in the medium (W)

W=I

2

Rt

I----current intensity R——resistance T-----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 effects:

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Ethyl violet 2390-59-2

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The sample and buffer ion diffusion rate increases, causing the sample separation band to widen:

2

Produce convection, causing mixing of the objects to be separated:

3

If the sample is sensitive to heat, it can cause protein denaturation:

4

Causes annual media reduction, resistance drop, and so on. The heat generated in electrophoresis is usually emitted from the center to the outer periphery, so the center temperature of the medium is generally higher than the outer circumference, especially the tubular electrophoresis, thereby causing the viscosity of the central portion of the medium to decrease relative to the peripheral portion, the friction factor is reduced, and the electrophoretic migration is performed. The speed is increased, and since the electrophoresis speed of the central portion is faster than the edge, the electrophoretic separation band is usually called an arch. Reducing the current intensity can reduce the heat generation, but it will prolong the electrophoresis time, causing the increase of the diffusion of the biological macromolecule to be separated and affecting the separation effect. Therefore, the appropriate electric field strength should be selected in the electrophoresis experiment, and the temperature can be appropriately lowered to obtain a better temperature. seperate effect.

4

[Biological reagents:

Ethyl violet 2390-59-2

]

Electroosmosis; the relative movement of a liquid in an electric field to a solid support medium. Since there may be some charged groups at the end of the support, such as the surface of the filter paper usually has some hydroxyl groups, the agar may contain some sulfate groups, and the surface of the glass usually has Si-OH groups. After ionization of these groups, the surface of the support medium is charged, and some oppositely charged ions are adsorbed, which move in the direction of the electrode under the action of the power plant to form a flow of the solution on the surface of the medium. This phenomenon is electroosmosis. When the pH is higher than 3, the surface of the glass is negatively charged, and the positive ions in the solution are adsorbed.

[Standard:

Ethyl violet 2390-59-2

]

The solution layer near the surface of the glass is positively charged, and under the action of the electric field, it migrates to the negative electrode, and the electrode liquid is caused to generate an electroosmotic solution to the negative electrode. If the direction of electroosmosis is the same as the direction of electrophoresis of the molecule to be separated, the electrophoresis speed is increased: if it is reversed, the electrophoresis speed is lowered.

5 Screening of the supporting medium: The size of the mesh of the supporting medium has a significant influence on the electrophoretic migration speed of the biomolecule to be separated. In the medium with large mesh openings, the surge speed is fast, and vice versa, the surge speed is slow.

[Biological reagents:

Ethyl violet 2390-59-2

]

Qingdao Jieshikang Biotechnology Co., Ltd. mainly operates: elisa kit, medium, standard and so on.