one operating

1 Pretreatment of cellulose acetate film

A gently draw a sample line with a pencil on the matte side of the diaphragm. The sample line can be selected at 2CM from one end of the diaphragm .

B. Place the electrode buffer in an incubator and carefully spread the membrane on the electrode buffer surface, usually with the matte side of the membrane facing down. The bottom surface of the diaphragm absorbs the electrode buffer and then gradually sinks until the electrode buffer completely immerses the membrane.

C After the membrane is soaked in the electrode buffer for a few minutes, it is taken out with a blunt tweezers and sandwiched between two layers of filter paper to absorb excess electrode buffer, but not overdried.

D is applied to the sample line of the matte side of the diaphragm, usually for linear loading, without dot loading. A serum protein sample "printed" was drawn on the sample line using a stamp ( 15 mm long and 3 mm wide plexiglass). When printing the cover, note that the distance between the ends of the stamp to the edge of the diaphragm is substantially equal.

The sample loading amount or sample loading volume of the E sample varies greatly depending on the concentration of the sample, the dyeing and the calibration method, etc. Usually , the amount of the protein sample added by 0.5 mg per sample line is insufficient. The amount can be reprinted once in the original position.

F. Place the sampled membrane on the electrophoresis cell holder with a blunt tweezers. The 10 mm portion of the diaphragm is in close contact with the top surface of the stent. The cellulose acetate membrane relies on its own adhesion in the electrode buffer. Will be tight on the membrane holder at both ends of the electrophoresis tank. Place several diaphragms in the same electrophoresis tank at the same time, and the adjacent diaphragms should be separated by more than 3mm .

Injecting the two electrodes G electrophoresis tank chamber a suitable amount of an electrode buffer, 40mm length with a filter paper for 2 to 3 layers bypass, one end of the filter paper is aligned with the leading edge of the bracket, the other end of the electrophoresis buffer tank invasion. After the filter paper strip is wet, remove the air bubbles so that the filter paper is in close contact with the bracket.

H Place the sampled membrane on the holder of the electrophoresis tank with a blunt tweezers. Add the sampled end to the negative end bracket and the other end to the positive end bracket.

I Cover the electrophoresis tank cover and let the membrane stand horizontally in the electrophoresis tank for 10 minutes. Then, the two electrodes of the electrophoresis tank are respectively connected to the positive and negative electrodes of the electrophoresis DC power supply.

J power on the power supply allows the electrophoresis instrument to operate in a regulated state, and the voltage is adjusted to 120~160V , which is equivalent to an electric field strength of 15~20V/cm . It can also make the electrophoresis instrument work in a steady state, and the current intensity depends on the number of diaphragms. Generally, the current intensity per membrane is controlled to be 0.4 to 0.8 mA . The electrophoresis time is 50~70min .

K cellulose acetate membrane electrophoresis is usually carried out at room temperature. Increase the electric field strength to speed up the electrophoresis. However, the higher the amount of heat generated in the membrane per unit time, resulting in a large amount of evaporation of the buffer, so effective cooling is required to avoid drying of the membrane. Tap water is usually placed in the cooling tank, and the cold brine and ice cubes are continuously cooled.

L electrophoresis is completed.

M immersed the membrane.

N considerations

1 Pretreatment before membrane electrophoresis

  The cellulose acetate membrane has a lower affinity for water than paper, and it is necessary to ensure that the membrane contains a certain amount of buffer during electrophoresis, and the membrane must be previously impregnated in the buffer. The correct method of immersing the membrane should be to carefully spread the membrane over the surface of the buffer, relying on the bottom surface of the membrane to gradually absorb the buffer and then sink into the buffer. If the membrane is completely immersed from the beginning, many small air bubbles will accumulate on the membrane to form many opaque spots, which takes a long time to saturate the membrane, which not only wastes time but also affects the subsequent separation effect. In addition, when the membrane is saturated, the excess buffer is removed by the filter paper, and it is neither too dry nor too wet. Too dry is not conducive to electrophoresis separation; too wet, will affect the sample loading, widening the sample line, and the starting point of each component during electrophoresis will be uneven, thus affecting the separation effect.

2 electrode buffer concentration selection

A commonly used pH 8.6 barbital buffer is generally selected at a concentration between 0.05 mol/L and 0.09 mol/L . When selecting, first set a certain concentration first. For example, the length of the strip between the two poles in the electrophoresis tank is 8~10cm , the voltage per cm is required to have a voltage of 25V , and the current intensity is required to be 0.4~0.5mA per cm . If this value is not reached or exceeded during electrophoresis, the buffer concentration should be increased or diluted. If the buffer concentration is too low, the effect is that the zone moves too fast and the zone width increases. If the buffer concentration is too high, the zone moves too slowly, which causes some separation zones to be too concentrated and difficult to distinguish. . It is important to note that in cellulose acetate membrane electrophoresis, since most of the current is conducted by the sample, a lot of heat is generated. Sometimes the selected buffer concentration is considered to be suitable, but in the case of an increase in ambient temperature or a higher voltage, the thermal evaporation of water is exacerbated, which can also cause the buffer concentration to be too high, even making the diaphragm The consequences of cognac.

    Note: Acetate diaphragm size is 70 * 90

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