JPS63271154A - High separability electrophoresis method - Google Patents

Abstract

PURPOSE:To increase the separability of a gel electrophoresis method by increasing the thickness of a gel in the migration direction. CONSTITUTION:A gel plate increased in the thickness of the gel in the migration direction is used in the electrophoresis method using a gel-like material as a medium for sepn. Namely, the gel having such as shape of which the cross section cut at the plane parallel with the migration direction is as shown in the figure is used. A more concrete example thereof is such a gel plate of which the gel thickness increases exponential-functionally in the migration direction or gel plate of which the gel thickness increases linearly in the migration direction and the gel thickness at the end point of the migration is (1+2<1/2>) times the gel thickness at the start point of the migration.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for separating and analyzing proteins, nucleic acids, etc., and particularly to an electrophoresis method suitable for separating components of these substances with similar molecular weights.

[Conventional technology]

As is well known, the migration distance of a charged molecule placed in an aqueous solution, y, is the electric field strength applied to the solution, E, and the migration time, t, as shown in the following equation (1).

Assuming the migration speed per unit time and unit electric field strength to be 70, y=uoEt...
...It is given by (1). In a gel-like medium, the migration distance decreases from the value given by equation (1). When using a gel plate of constant thickness, Proceedings of the National Academy of Sciences, Vol. 65, No. 9
70 pages or less, 1970 (ProcNat' Q Ac
ad Sci, 65.970-970 (1970)
) are discussed.

[Problem that the invention seeks to solve]

Since the electric field strength in the gel is constant (E) throughout the gel, y
is given by the following equation.

y = uoE te - (2) Here, is a parameter that has a positive correlation with the molecular weight of the migrating molecules, and T is the gel concentration. In addition, it is known that in denatured nucleic acid and protein molecules, 0 is a constant value independent of the molecular weight.

According to equation (2), molecules of different sizes have different k, resulting in different y, and even if a mixture is electrophoresed, they will be separated from each other on the gel. The limit of the resolution of this conventional technology is 1
The k of the two molecules A and B that we are trying to separate is ^,
ka, the maximum separation ratio of these two components is 1lt(Δ
y, 1).

Generally, the separation distance Δy of the above two components when the gel concentration is T,
is Δy = u oE t (e −kA” −e″″k
BT) , , , , (3). T of Δy
By setting the differential coefficient with respect to zero as zero, the value of T (T,) that maximizes Δy can be determined as follows. In other words, the gel concentration suitable for separating two components with close molecular weights is the limit value of T1 when ^→ka (=k), that is, 1
/ given as. Then, when T=1/, the Δy of the A and B components when the small component A migrates, that is, Δy, 1 is Δymz=L(1-e'-1)
−(5). Here, R is a positive number smaller than 1 and equal to ^/ka. When R = 1, of course Δy = O. Since R is close to 1, from the differentiation with respect to R on the right side of equation (5), the minute displacement dyz (absolute value) of y for the minute displacement dR of R from 1 is dyz=LdR
・・・・・・・・・(6) can be written. That is, in the prior art, separation can be achieved by L times dR corresponding to the molecular weight difference between the two components, which is the maximum separation distance.

Based on the above principle, it is an object of the present invention to provide an electrophoresis method that provides a separation distance of L times or more of a constant dR, whereas the prior art allows separation only by L times a constant dR.

[Means for solving problems]

The above object is achieved by increasing the thickness of the gel in the direction of migration. That is, a gel whose cross section cut along a plane parallel to the electrophoresis direction has a shape as shown in FIG. 1 is used. In Figure 1, 1 is a cross section of the gel, 2
is the upper surface of the gel, which is generally a curved surface. The gel had a length L in the direction of migration, and the bottom surface was flat for ease of handling.

[Effect]

If the thickness of the gel is expressed as a function f (y) of the migration distance y, the total resistance ρ0 of the gel will be given by Ohm's law. Here, γ is the specific resistance of the gel, and the width of the gel (depth in the direction perpendicular to the electrophoresis direction) is the unit length. The total voltage difference applied to the gel is Eo
Then, the electric field strength d E/ at the migration distance y of the gel is
d y becomes, and the migration speed in this gel is d y/d t
Hato becomes. If f(y) is specified specifically, the differential equation (
9), y as a function of t is found.

Now, consider a gel in which the gel layer rapidly increases exponentially. The gel thickness at the starting point of migration is set as J' (y) = CD' = Ce"nD" - (10) where C and D are real numbers larger than 1. In this case, as a solution to equation (9)...
11) is obtained. Using the same method used to obtain equation (4), T in this case becomes...(12). At this T, Δy when migrating to the pole when ^→kn (=k), that is, Δy, 2 is...
...(13) becomes. By differentiating ΔymZ with R and finding the limit value of R→1, the separation power dyz of the exponential gel corresponding to equation (6) is dyz = -CD' -1) d R...
...(14)nD. If we calculate the ratio of the absolute values of dy from equations (14) and (6), we get: dyt 2 6 ...... (15) dyz is larger than dyz, and the exponential gel has a more uniform thickness. It can be seen that the resolution is higher than that of gel.

Next, the separation power of a gel whose thickness increases linearly, which is easier to produce than an exponential gel, is determined. In this case, if the gel thickness at the starting point is s and the thickness at the end point is a, then the following equation is obtained. In the same way as above, we can calculate the limit value of T when ＾→ka (=k), so that ・・・・・・・・・(17) ・・・・・・・・・(18) The separation distance, Δy, 8 is (19). By differentiating Δy1δ with R and setting R=1, the resolution dys of the linear gradient gel is obtained as follows: where g is the ratio of the thickness at both ends of the gel, that is, g=a/
It is b. As can be seen by differentiating dys with respect to g, c
tyδ has a maximum value (approximately 1.21 LdR) larger than LdR at g = 1 + 5.

Furthermore, as can be seen from equation (14), even if L is constant, D
By increasing , the separation power of the exponential gel increases as much as possible.

Furthermore, if the high-resolution gel of the present invention is combined with another separation method, such as one based on isoelectric point difference, a two-dimensional electrophoresis method with high resolution becomes possible.

As described above, in the present invention, by setting the electric field strength to gradually decrease with the migration distance, it is possible to increase the difference in the migration distance between the two components.

〔Example〕

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 Purification was determined in advance by a conventional method and the molecular weight was 68340.
A mixture of single-stranded deoxyribonucleic acids (labeled with 82P) known to be 68,000 and 68,000 was used as the sample to be analyzed.

First, this sample was mixed with a gel having a uniform thickness of 0.5 mm and a length of 10 am (gel concentration 4.6%, 7M urea, 90%
Electrophoresis was performed using an mM Tris-boric acid aqueous solution, pH 8, 3° containing 1 mM ethylenediaminetetraacetic acid. After electrophoresis, an X-ray film was placed on the gel and an autoradiogram was created using lap beta rays. When this autoradiogram was examined, even when the sample was migrated 10 cm to the edge of the gel, there was only one separated band, and no separation of the two components was observed.

On the other hand, the same sample has the same gel length, but a gel whose thickness increases exponentially so that the thickness is 0,5 r @ at the migration start point and 2.5 ma at the end point (concentration 23%, bottom surface is flat, (created using the al type).

When I created an autoradiogram, the fast component was l0
Two separated bands were clearly recognized at the time of GI electrophoresis. The distance between the two separated bands was approximately 1.0 m.

Example 2 An autoradiogram was created by electrophoresing the same nucleic acid sample as above using a gel (concentration 4.6%) with a uniform thickness of 0.5 mm and a length of 70 countries. When I checked it after electrophoresis, the distance between the two bands was about 2.0.
It was mm. On the other hand, the same nucleic acid sample was run on a linear thickness gradient gel (gel concentration 7.9%, length 70 mm) with a thickness of 0.5 mm at the starting point and a thickness of 1.2 rrtn at the end point.
When the fast component migrated at 70G, the distance between the two separated bands was expanded to approximately 3.0 m.

〔Effect of the invention〕

According to the present invention, since the separation power of gel electrophoresis is increased, the efficiency of determining the base sequence of a nucleic acid can be increased. It also has the effect of allowing more accurate evaluation of the purity of purified protein samples.

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view of a gel used in an example of the present invention.

Claims (1)

[Claims] 1. High separation characterized by using a gel plate in which the gel thickness increases exponentially in the direction of electrophoresis in an electrophoretic separation method using a gel-like substance as a separation medium. electrophoresis method. 2. In an electrophoretic separation method using a gel-like substance as a separation medium, the thickness of the gel increases linearly in the direction of migration, and the thickness of the gel at the end of migration is equal to that of the gel at the start of migration. A high-resolution electrophoresis method characterized by using a gel plate having a thickness (1+√2) times.