JP3838449B2 - Second dimension standard line forming device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a second-dimensional standard line forming apparatus. More specifically, the present invention is used as a means for separating and identifying proteins in the fields of biotechnology and pharmaceutical industry, measuring molecular weight and isoelectric point, determining protein names, and further determining protein purity. The present invention relates to a second-dimensional standard line forming apparatus useful for two-dimensional electrophoresis.
[0002]
[Prior art and its problems]
Conventionally, as a means of separating and identifying mixed proteins into individual component proteins, in a polymer gel plate or the like, a sample is first developed in one direction (X direction) with respect to the isoelectric point, and then in another direction (Y direction) with respect to molecular weight. The two-dimensional electrophoresis method (2-DE) developed in (1) is known. In this two-dimensional electrophoresis method, for example, when a two-dimensional gel plate is 20 cm × 20 cm, about 5000 types of proteins can be separated, but when the size is 30 cm × 30 cm, about 15,000 types, which is about three times as large as that. It is said that it is possible to isolate the protein.
[0003]
Recently, a gradient gel fixed to the first-dimension isoelectric focusing is commercially available, and the reproducibility of isoelectric focusing is increased, and the reproducibility of 0.1 pH unit is being secured. Furthermore, in the technique of electrically plotting the spot on the gel plate developed two-dimensionally on the PVDF membrane and analyzing the amino (N) terminal sequence of each protein, high sensitivity at the pmol (picomolar) level. It has become possible to identify proteins by searching and collating the analysis results obtained by this analysis method with a database in which the amino acid sequence information of proteins has been accumulated.
[0004]
On the other hand, due to the dramatic improvement in the performance of image analysis devices and computers, analysis by two-dimensional electrophoresis has been taken up as a project to be performed in a complementary or complementary manner to genome analysis of genes that have recently become popular. The two-dimensional electrophoresis project has been put into practice as a Proteome project, and is now in the limelight as almost the only means for analyzing the results expressed in vivo.
[0005]
However, in the conventional two-dimensional electrophoresis method, the standard protein has been used in many cases in each system, but the scale and form of the two-dimensional electrophoresis method varies depending on the apparatus used. Therefore, it was difficult to exchange two-dimensional electrophoresis data of different systems with each other. In addition, when analyzing diversified or time-varying proteins from different tissues to integrate and analyze all the proteins of the same organism, a series of standard proteins are used to analyze each of these two dimensions. The need to standardize and integrate electrophoresis screens has arisen.
[0006]
Therefore, the inventors of the present invention select in advance a group of extrapolated proteins that are moderately distributed on the two-dimensional electrophoresis screen in order to integrate and standardize many two-dimensional electrophoresis screens, and select these samples as samples. A method of co-electrophoresis with proteins has been proposed.
However, in these conventional two-dimensional electrophoresis methods, standardization was performed based on spots formed by a few standard proteins of several to several tens, so polyacrylamide gel plates used for two-dimensional electrophoresis There are problems such as being susceptible to local disturbances of electrophoresis due to distortion of the substance itself, and also due to distortion caused by substances mixed on the sample side such as salt, denaturant, coexisting organic and inorganic substances.
[0007]
The present invention has been made in view of the circumstances as described above, and relates to a two-dimensional electrophoresis method capable of solving conventional problems and improving the resolution and reliability of the two-dimensional electrophoresis method. It aims to provide new technical means.
[0008]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present invention provides a second-dimensional second-direction standard line in a two-dimensional electrophoresis apparatus in which a gel plate and a fixed isoelectric focusing gel plate at the upper end thereof are arranged between opposing glass plates. A forming apparatus, a setting latch disposed in the through-hole portion in the second dimension direction with a vertical groove as a latch surface and facing the inner surface, and a marker cup mounted on the setting latch so that the position can be selected. The marker cup has a cup body in which an extrapolated standard substance for second dimension electrophoresis is placed in the inner space, a lowermost capillary connected to the cup body, and a latch surface longitudinal groove on the setting latch. IPG (fixed) that has already been developed in the first dimension (X direction) during electrophoresis in the second dimension (Y direction). Isoelectric point The marker cup is placed at a predetermined position on the moving gel plate), and an extrapolated standard substance for the second dimension (Y direction) migration in the cup body is continuously added through a capillary tube and co-migrated with the sample. Thus, a second-dimensional direction standard line forming apparatus is provided that forms a linear standard line at a required interval in the second-dimensional direction (Y direction).
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Conventionally, the means for introducing the standard line of the first dimension (X direction) in the two-dimensional electrophoresis method does not require any special apparatus, and an IPG (immobilized isoelectric focusing gel which is placed at the upper end of the X direction. The plate) may be evenly immersed in the mixed solution of standardizing substances used for the X-direction standard line, or it may be added uniformly. However, in order to form a standard line in the second dimension (Y direction), it is almost impossible to manually add for a long time during electrophoresis, and if possible, the number of axes is limited. There were many examples that moved and failed.
[0010]
On the other hand, in the present invention, IPG (immobilized isoelectric) in which the development of the first dimension (X direction) has been completed for the extrapolated standard substance for Y-direction migration equal to or less than that of the standard protein. It is possible to continuously form a standard line of the second dimension (Y direction) by adding it to a predetermined position of the gel plate for point migration), and the first dimension (X direction) and the second dimension. In the dimension (Y direction), a standard line that is a linear reference for reading the molecular weight and isoelectric point can be introduced.
[0011]
Furthermore, in the present invention, since they can be shown as linear standard lines, the amount of information for standardizing local or global disturbances in two-dimensional electrophoresis is greatly increased.
In the following, embodiments of the present invention will be described in more detail with respect to the present invention in which such excellent actions are obtained by showing examples.
[0012]
【Example】
1, FIG. 2, and FIG. 3 of the attached drawings are a perspective view, a plan view, and a cross-sectional view taken along line AA, showing the second-dimensional standard line forming device of the present invention.
As shown in these drawings, this apparatus is mainly constituted by a marker cup (1) and a setting latch (2).
[0013]
The marker cup (1) has a cup body (11) having an inner space, a capillary tube (12) communicating with the inner space, and a guide rail portion (13). At the lower center of the cup body (11), A capillary tube (12) fitted with a gasket (14) made of silicon rubber or the like is attached to the outer periphery thereof, and this can be adjusted by about ± 5 mm in the vertical direction. In addition, the marker cup (1) is provided to face the inner surface of the through hole (21) of the setting latch (2) by the guide rail portion (13) disposed immediately below the cup body (11). The latch surface (22) is detachably guided and inserted into an arbitrary position of the longitudinal groove (23). At this time, for example, it is possible to move and install by 1 mm from the interval of the longitudinal groove (23) of the setting latch (2). However, by reducing the interval of this groove, the movement interval can be reduced as much as possible. Is possible.
[0014]
Also, a plurality of marker cups (1) can be installed on the setting latch (2). In this case, the installation interval between the cup bodies (11) is usually about 14 mm, but the shape of the cup body (11) By selecting (rectangular) and the material, for example, the distance can be made close to 5 mm. Further, it can be installed along a setting latch (2) having an electrophoretic width of the second dimension, for example, 200 mm (can be 100 to 500 mm depending on the length of the electrophoretic width).
[0015]
In the above apparatus, the IPG gel plate (5) is installed on the upper end of the SDS-polyacrylamide gel plate (4) sandwiched between the two opposing glass plates (3).
Specifically, for example, when the migration distance in the second dimension is 20 cm, an extrapolated standard substance for Y-direction electrophoresis dissolved in 200 μl of electrophoresis buffer (containing 5% glycerin), For example, add insulin. In this case, insulin is obtained by iminating an amino group and fluorescently labeling the hydroxyl group. The capillary tube (12) disposed immediately below the cup body (11) is adjusted and installed so as to contact the upper surface of the IPG gel plate (5) (18 cm) after completion of the first migration. Furthermore, everything is covered with a buffer solution to enable energization. The reason why glycerin is added to the electrophoresis buffer in the cup is that it is not mixed with the buffer.
[0016]
The marker cup (1) of this device is installed in several places to 10 to 30 sets with respect to the setting latch (2). By energization, several to 10-30 standard lines in the Y direction can be drawn. The interval is equidistant according to the above installation, or experimentally shown at the place where the isoelectric point of the standard substance group in the X direction of the one-dimensional isoelectric focusing and the isoelectric focusing location according to PI. It is possible to form them at predetermined intervals.
[0017]
As an addition method, as illustrated in FIG. 4, when added to the upper end at equal intervals, there is a case where nonuniform distribution is performed at intervals corresponding to the pI of the standard protein as shown in FIG. 5. In the case of equal intervals, it is sufficient to add them at equal intervals. On the other hand, when the Y axis is formed at intervals according to pI, one-dimensional IPG isoelectric focusing is performed for fluorescence for the X axis. Marker cup (1) as an applicator for the Y axis just above the migration distance by co-electrophoresing with a sample protein with a small amount of modified standard protein and observing the pI value of the standard protein from the moving point of the fluorescent protein. Place two-dimensional electrophoresis.
[0018]
In fact, in FIGS. 4 and 5, the linearity of the standard line in the second dimension (Y direction) indicates the state of electrophoresis. That is, in the case of a vertical straight line, it indicates that there is no distortion or local bias in the result of electrophoresis, and the distortion is the salt content contained in the SDS-polyacrylamide gel plate (4) and the sample protein. Shows the distortion of the electrophoretic state caused by. Therefore, the standard line in the Y direction can be captured on the graphic by the detector, and thereby the result of spot migration can be standardized.
[0019]
4 and 5 illustrate the obtained standard lines.
[0020]
【The invention's effect】
According to the present invention, as explained in detail above, the extrapolated standard substance for migration in the second dimension (Y direction) is an IPG (immobilized isoelectric point) in which the development in the first dimension (X direction) is completed By adding it to a predetermined position of the electrophoresis gel plate), it becomes possible to continuously form a standard line of the second dimension (Y direction), the first dimension (X direction) and the second dimension. A standard line, which is a linear reference for reading the molecular weight and isoelectric point, can be introduced into the eye (Y direction). Furthermore, since they can be shown as linear standard lines, accurate information for standardizing local or global disturbances in two-dimensional electrophoresis is obtained, and the amount of information is greatly increased.
[0021]
As a result, the resolution and reliability of the two-dimensional electrophoresis method can be improved, and thus it is considered that it contributes widely to the fields of biotechnology and pharmaceutical industry that require protein separation and identification.
[Brief description of the drawings]
FIG. 1 is a perspective view showing a second-dimensional standard line forming apparatus of the present invention.
FIG. 2 is a plan view corresponding to FIG. 1;
3 is a cross-sectional view taken along the line AA in FIG.
FIG. 4 is a diagram illustrating a standard line obtained when added at regular intervals.
FIG. 5 is a diagram exemplifying a standard line obtained in the case of non-uniform distribution at intervals corresponding to pI of a standard protein.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Marker cup 11 Cup main body 12 Capillary tube 13 Guide rail 14 Gasket 2 Setting latch 21 Through-hole part 22 Latch surface 23 Longitudinal groove 3 Glass plate 4 SDS-polyacrylamide gel plate 5 IPG gel plate

Claims (1)

A second-dimension direction standard line forming device in a two-dimensional electrophoresis apparatus in which a gel plate and a fixed isoelectric focusing gel plate at the upper end thereof are arranged between opposing glass plates, and penetrating in the second dimension direction. There is a setting latch that is provided with a longitudinal groove as a latching surface in the hole and is opposed to the inner surface thereof, and a marker cup that is mounted on the setting latch so that the position can be freely selected. A cup main body for inserting an extrapolation standard substance for dimensional eye migration, a capillary tube at the lowermost end communicating with the cup body, and a guide rail portion that is detachably inserted in the longitudinal groove on the latch surface of the setting latch between the two. The marker cup is placed at a predetermined position on the immobilized isoelectric focusing gel plate that has already been developed in the first dimension during the second dimension electrophoresis, and the inside of the cup body second A linear standard line is formed at a required interval in the direction of the second dimension by continuously adding an extrapolation standard substance for original eye movement through a capillary tube and co-migrating with a sample. Dimensional direction standard line forming device.

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