JPS6126848A - Two-dimensional electrophoresis device - Google Patents

Abstract

PURPOSE:To measure migration spots with high precision by adding comparison samples in a flat plate gel after secondary electrophoresis and generating the calibration line between the dye concentration and weight of the comparison samples fo every secondary gell. CONSTITUTION:Plural comparison samples 6 whose weight is alreadly knwon as well as an object of detection are added in the gell 5 after secondary electrophoresis. Each migration spot 12 of the object of detection separated in the secondary gel 5 and each comparison sample 6 are dyed in silver at the same time and dye concentration values of both objects are measured. Then, a calibration line which decreases in dye concentration proportionally; from light weight to heavy weight is geneated only for the comparison sample 6. Then, the weight corresponding to dye concentration on the calibration line corresponding to the dye concentration of each migration spot 12 of the object of detection is measured by calculation.

Description

DETAILED DESCRIPTION OF THE INVENTION (A) Field of Industrial Application This invention relates to a two-dimensional electrophoresis apparatus, and more particularly to a two-dimensional electrophoresis apparatus that can improve the accuracy of quantitative determination of a subject.

(B) Prior art The weight of the test object in the sample in the conventional two-dimensional electrophoresis apparatus is measured by silver staining the electrophoresis spot of the test object separated in the flat gel after the completion of the second-dimensional electrophoresis. This was done by staining with a coomalet blue method or the like and measuring the intensity of the staining optically, for example by an absorption method, and calculating the optical information obtained by a computer into quantitative information.

However, with this method, if the staining concentration of the electrophoresis spot to be tested differs depending on the loft of the test sample, or if the second-dimensional gel, staining reagent, temperature during staining, etc. Even if the spots had the same weight, their staining concentrations were different, reducing the quantitative accuracy of each electrophoresis spot.

(c) Purpose This invention was made in view of the above circumstances, and one of its main purposes is to prepare a comparison sample, which is the same as the test object and whose weight is known, in the flat gel after the second-dimensional electrophoresis. , and create a calibration curve between the staining concentration and weight of the comparison sample for each second-dimensional gel. The purpose is to enable quantitative measurement of spots with high precision.

(D) Structure This invention provides a second-dimensional gel, a second-dimensional electrophoretic separation means for electrophoretically separating the test object of the test sample after the first-dimensional electrophoresis in the second-dimensional direction of the gel. , after the completion of the second-dimensional electrophoretic separation, a plurality of comparative samples of different weights, which are the same as the test object and whose weight is known, are placed in a second-dimensional gel in either the second-dimensional separation direction or the first-dimensional separation direction. An addition means for adding in a spot to one or both of them, a staining intensity measuring means for staining the comparison sample and the test object and measuring the staining concentration of both, and a staining intensity output from this staining intensity measuring means. A creation storage means for creating and storing a calibration curve between the weight and staining concentration of each comparison sample based on the signal, and calculating the weight of each electrophoresis spot of the subject based on the storage signal output from this creation storage means. This is a two-dimensional electrophoresis device equipped with a calculation means to perform the following steps.

(e) Examples The present invention will be described in detail below based on examples shown in the drawings. Note that this invention is not limited to this.

In FIG. 1, a two-dimensional electrophoresis apparatus (11) is mainly composed of a creation storage means (2), a calculation means (3), and an addition means (4).

The creation storage means (2) and calculation means (3) consist of a microcomputer (C), and the second-dimensional rectangular plate gel (5)
Light is emitted from above, and a staining degree measuring means (9) receives the photometric (8) that has passed through the stained sample (61 (71), which will be described later) in the gel (5) and measures the staining concentration of the sample.
) is electrically connected to. This measurement means includes a light emitting section (not shown), a light receiving section αO) and a photometric signal processing section (11
) consists of a densitometer.

The creation storage means (2) creates a calibration curve between the weight and staining concentration of the sample (61 (of 71), a comparison sample (6) whose weight is known) based on the staining intensity signal output from the measurement means (9). On the other hand, the calculation means (3)
is configured to calculate the weight of a test sample (7) of unknown weight among the samples (6) and (7) based on the storage signal output from the creation storage means (2) and the staining intensity signal. There is.

The addition means (4) consists of a pipette, a syringe, etc.

Next, in the above device, the test object in the test sample (7),
For example, a method for quantitatively measuring the weight of proteins in serum will be explained.

First, as shown in FIG. 2, a plurality of comparison samples (6), which are the same as the test object and whose weights are known, are added in spots into the gel (5) after the completion of second-dimensional electrophoresis.

These comparison samples (6) have different weights, for example, from 10-tog to 10-6g, and the weight is increased in descending order of weight toward the molecular weight level of the second-dimensional gel (5) (in the two-dimensional direction). They are lined up. Then, each electrophoresis spot (12) of the test object separated in the second-dimensional gel (5) and each comparison sample (6) are simultaneously silver-stained.
□Measure the staining density of both of these. Next, a calibration curve (A) is created for only comparative sample (6) in which the staining density decreases proportionally from the lighter weight to the heavier weight (see Figure 3). Then, the staining concentration corresponding to the staining concentration of each electrophoresis spot (12) of the subject to be tested is taken on the calibration curve (A), and the weight corresponding to this is calculated and measured.

Alternatively, the comparative sample (6) may be added to the second-dimensional gel (5) in the charge level direction (-dimensional direction) as shown in FIG. In this case, a calibration curve (B) as indicated by the broken line in FIG. 3 is created in the same manner as above, and the weight of the subject to be tested is measured.

Furthermore, if it is possible that the slope of the calibration curve changes depending on the position where comparative sample (6) is added,
) is added not near the periphery of the second-dimensional gel (5) as shown in Figure 2, but near each electrophoresis spot (12) of the subject in the - and two-dimensional directions of the gel (5). You can go there.

By creating a calibration curve every time the second-dimensional gel is exchanged using the comparison sample as described above, and quantitatively measuring each electrophoresis spot of the test target, it is possible to confirm that the staining concentration differs from one lot of the test sample to another. Even if the second-dimensional gel, staining temperature, staining reagent, etc. change, the staining concentration can be measured under the same conditions, improving the quantitative accuracy of each electrophoresis spot to be tested. can.

(f) Effect This invention adds a plurality of comparison samples whose weights are known to a second-dimensional flat gel in either one-dimensional direction or two-dimensional direction, and calculates the weights and staining concentrations of these comparative samples. A calibration curve was created, and the weight of each electrophoresis spot of the test object was calculated based on this calibration curve. Therefore, even if the staining concentration between test sample lofts differs, the weight of each electrophoresis spot of the test object can be calculated based on this calibration curve. can be quantitatively measured with high precision, and each electrophoresis spot can be quantitatively measured with the same accuracy even if the second-dimensional gel, staining reagent, temperature during staining, etc. change.

[Brief explanation of drawings]

Fig. 1 is an explanatory diagram of the main part configuration showing one embodiment of the two-dimensional electrophoresis device according to the present invention, Fig. 2 is a perspective view of the second-dimensional gel showing the addition state of this comparative sample, and Fig. 3 is this calibration diagram. It is a line graph. (1) - Two-dimensional electrophoresis device, (21- Creation storage means, T3) - Calculation means, (4) - Addition means, f5) - Second-dimensional gel, f6) - Comparative sample, (7) - Test sample, (
91-Staining intensity measuring means, (12)-Migration spot of test object, (A) (B)--Calibration curve.・−”j

Claims (1)

[Claims] 1. a second-dimensional gel, a second-dimensional electrophoretic separation means for electrophoretically separating the test object of the test sample after the first-dimensional electrophoresis in the second-dimensional direction of the gel; After the second-dimensional electrophoretic separation is completed, multiple comparison samples of the same but different weights with known weights are placed in the second-dimensional gel in either the second-dimensional separation direction or the first-dimensional separation direction, or both. an addition means for adding in spots, a staining intensity measuring means for staining the comparison sample and the test subject and measuring the staining concentration of both, and a staining intensity signal outputted from this staining intensity measuring means for each comparison. A preparation storage means for preparing and storing a calibration curve between the weight of the sample and the staining concentration, and a calculation means for calculating the weight of each electrophoresis spot of the test object based on a storage signal output from the preparation storage means. A two-dimensional electrophoresis device.