JPS61221389A - Method and apparatus for electrophoresis - Google Patents

Abstract

PURPOSE:To separate a sample having a wide mol.wt. distribution with one time of separating operation in a short period by increasing continuously or stepwise the voltage to be impressed to both terminals of a migration zone. CONSTITUTION:An upper electrode 10a in buffer soln. cells 1 for migration provided at both ends of a migration pipe 2 is connected to the negative side of a DC power source 11 for automatic voltage increase and a lower electrode 10b is connected to the positive side. The sample soln. is injected between the upper part of the tube 2 and the cells 1 to form a sample zone. The power source 11 is operated to impress the voltage to the electrodes. The voltage is continuously or stepwise increased as time passes by. The sample having the wide mol.wt. distribution including high mol.wt. is thoroughly separated with the single separating operation by the above-mentioned method.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an electrophoresis method and apparatus used for separating and purifying proteins, nucleic acids, etc.

[Conventional technology]

As an electrophoresis method, for example, a zone electrophoresis method using starch gel, acrylamide gel, etc. as a support is known. In this method, the support that is poured into the electrophoresis tank together with the buffer solution is gelled and used as the electrophoresis phase, and the components in the sample are not only separated due to differences in mobility, but also molecules in the gel eyelids. Since it is also separated by a sieving effect, it has a good separation effect and is effective as a means for analysis or separation and purification of various samples.

However, for samples with a wide molecular weight distribution, separation is insufficient and takes a long time.In particular, for samples with a wide molecular weight distribution including those with high molecular weights, it is necessary to separate the samples several times by changing the gel concentration. There was the trouble of having to perform a separation operation. In order to solve this problem, it is conceivable to shorten the separation time by increasing the voltage, for example, but in this case, the problem arises that low molecular weight substances cannot be sufficiently separated.

[Problem that the invention seeks to solve]

The present invention has been made in view of the above circumstances, and its purpose is to provide an electrophoresis method and method that can separate even samples with a wide molecular weight distribution, including those with high molecular weight, in a single separation operation. The purpose is to provide such equipment.

[Means for solving problems]

In order to solve the above problem, the present inventor conducted intensive research focusing on the voltage applied to the support, and found that by increasing the applied voltage over time, samples with a wide molecular weight distribution including those with high molecular weight However, the present inventors have discovered that even in the case of a single separation operation, the separation can be carried out satisfactorily, and the present invention has been completed.

That is, the present invention is characterized in that the voltage applied to both ends of the migration zone is increased continuously or stepwise over time.

It is also characterized by being equipped with a migration zone equipped with voltage application means that increases the voltage continuously or stepwise over time, and a detector that detects the sample separated in the migration zone. .

〔Example〕 An embodiment of the present invention will be described below with reference to the drawings.

FIG. 1 shows an example of the apparatus of the present invention. Code 1 in the diagram
is a buffer tank for running, 2 is a migration tube, 3 is a running gel, 4 is a buffer tank for elution, 5 is a pump, and 6 is a detector (ultraviolet photometer).
, 7 is a recorder, 8 is a fraction collector, 9 is a semipermeable membrane, 10a and 10b are electrodes, and 11 is an automatic voltage step-up DC power supply. It is.

The migration buffer tank l is provided at both ends of the migration tube 2,
It is filled with a phosphate buffer containing 0.1% sodium dodecyl sulfate (SDS). The electrophoresis tube 2 is filled with a electrophoresis gel 2 containing, for example, 10% acrylamide, 2.7% NN'-methylenebisacrylamide, and 30°1% SD.

The elution buffer tank 4 is filled with the same buffer as the electrophoresis buffer tank 1, and the buffer is pumped by a pump 5 to an elution section 12 provided with a semipermeable membrane 9, a detector 6, and a fraction collector 8. sent to.

The recorder 7 records the relationship between the voltage application time and the absorbance of the component detected by the detector 6.

The upper electrode 10a is connected to one side of the automatic voltage step-up DC power supply 11, and the lower electrode 10b is connected to the + side. The automatic voltage step-up DC power supply 11 is composed of a rectifying section, a variable resistance section provided on the output side of the rectifying section, and a moving section that moves the slider of the variable resistor. At the same time, the applied voltage is increased continuously or stepwise.

Next, an example of the electrophoresis method of the present invention will be explained using the above apparatus.

The sample was Bio-Rad's 5DS-PAG E 5, which is a mixture of six types of proteins with known molecular weights as shown below.
Standard L MW: Soybean Tribucin Inhibitor 21,500 Carbonic Anhydrase 31,000 Ovalbumin 45,000 Bovine Serum Albumin 66.200 Phosphorylase 8 92,500 with 20% Sucrose and 1
The sample is diluted 4 times with a 0.1M phosphate buffer having a pH of 7 containing %2-methylcaptoethanol and heated at 37°C for 2 hours before use.

This sample solution is 25μ! is injected between the upper part of the electrophoresis tube 2 and the electrophoresis buffer tank 1 to form a sample zone, and the automatic voltage step-up DC power supply 11 is operated to apply a voltage.

The rate of increase of this applied voltage is 5 V/hour and increases continuously (see FIG. 2).

Five hours after the start of voltage application, the components in the sample are sequentially separated and sent to a detector 6 to measure absorbance, and then collected into a fraction collector 8.

FIG. 3 shows the relationship between the voltage application time and the absorbance of the separated components. As is clear from the figure, it can be seen that components with a small molecular weight and components with a large molecular weight are separated by - times of separation operations. Figure 4 shows the same sample at a constant voltage (30V).
This shows the case of separation. In this case, the higher the molecular weight (phosphorylase B), the longer the separation time will be. For this reason, a sample containing a substance with a higher molecular weight cannot be separated by the second operation.

FIG. 5 shows the relationship between the peak time (retention time) appearing on the detector 6 and the molecular weight of the protein. According to the figure, the retention time increases linearly as the molecular weight increases.

On the other hand, in the conventional case (applied voltage constant), the retention time increases exponentially as the molecular weight increases, so it is clear that the method of the present invention shortens the retention time on the high molecular weight side. It is.

The following table shows the molecular weight and retention time of the proteins.

1 Emperor's 1 minute weight - Meng poem plate 1 invention Conventional example 14.400 5.0 3.821.500
5.4 4.231.000 5.8
4.845.000 6.6 5.766.
200 7.8 7.995.500
9.4 10.5 Figure 6 shows the separated components detected by detector 6.
Instead of sending the elution part 12 to the ultraviolet rays directly,
The transmitted light is detected by a detector 6.

In the above embodiment, an ultraviolet spectrometer was used as a detector for the separated components, but a visible spectrometer, a radiation detection device, etc. can be used. Also,
Although the case where it is applied to zone electrophoresis is shown, it is not limited to this, and in addition to methods using a support such as filter paper electrophoresis and continuous filter paper electrophoresis, electrophoresis that does not use a support (
Tiselius electrophoresis method, density gradient electrophoresis method, etc.)
It can also be applied to

〔Effect of the invention〕

As explained above, according to the present invention, the voltage applied to both ends of the migration zone is increased continuously or stepwise with the passage of time, so that a wide molecular weight distribution including high molecular weight molecules can be obtained in - times of separation operations. It is possible to separate samples that have Moreover, separation time can be shortened.

[Brief explanation of drawings]

Fig. 1 is a schematic diagram showing an embodiment of the electrophoresis device of the present invention, Fig. 2 is a waveform diagram of applied voltage, Fig. 3 is a graph showing the relationship between voltage application time and absorbance, and Fig. 4 is a graph showing the relationship between voltage application time and absorbance. FIG. 5 is a graph showing the relationship between voltage application time and absorbance in a conventional example, FIG. 5 is a graph showing the relationship between protein molecular weight and retention time, and FIG. 6 is a graph showing another embodiment of the electrophoresis apparatus of the present invention. This is a schematic diagram. DESCRIPTION OF SYMBOLS 1... Buffer tank for electrophoresis, 2... Migration tube, 3... Running gel, 4... Buffer tank for elution, 5... Pump, 6
...detector, 10a, 10b...electrode, 11...
Automatic voltage step-up DC power supply. Figure 1 (Re Figure 2 Time (hrs) Figure 3 ■ Figure 4 Figure 5 +!-i (MwX +04) Mold ejection 10b

Claims (2)

[Claims] (1) An electrophoresis method characterized by increasing the voltage applied to both ends of a migration zone continuously or stepwise over time. (2) The electrophoresis zone is equipped with a voltage application means that increases the voltage continuously or stepwise over time, and a detector that detects the sample separated in the electrophoresis zone. electrophoresis device.