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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for preparing a polyacrylamide
gel plate for use in electrophoresis, and, more particularly, to a process
for preparing a large quantity of polyacrylamide gel plates which are
especially suitable for electrophoresis analysis of in vivo components
with a high molecular weight, such as proteins and the like.
2. Description of the Background Art
Polyacrylamide gels have widely been used for electrophoresis analysis of
in vivo components with a high molecular weight, e.g., protein, nucleic
acid, and the like. Usually, these polyacrylamide gels are prepared by
cross-linking polymerization of a monomer and a cross-linking agent; i.e.,
by adding a polymerization initiator to a 2-40% by weight aqueous solution
comprising a monomer such as acrylamides and a divalent or polyvalent
cross-linking agent such as N,N'-methylenebisacrylamide (such a solution
is hereinafter sometimes referred to as an "acrylamide monomer solution").
As an initiation method of polymerization, either a combination of a
peroxide and a reducing agent, or a combination of a peroxide, a reducing
agent, a photosensitation agent and exciting ray, while the reducing agent
is not necessarily used, may be used. For the latter photopolymerization
method, although some methods are proposed, in which the polymerization
does not take place before the light irradiation, even after a
polymerization initiator added (e.g., Japanese Patent Laid-open (kokai)
No. 91849/1987), the photopolymerization method basically involves many
technical problems for obtaining stable gel products with a good
reproducibility. The problems are found in difficulties in controlling the
polymerization reaction such as shading of the reaction liquid for gel
formation, selection of irradiation conditions for producing gel, and
occurrence of continued polymerization in the presence of light even after
the completion of gel formation. For these reasons, the former chemical
polymerization method, which adopts a combination of a peroxide and a
reducing agent, is preferred owing to its comparatively easy handling of
the reaction liquid and capability of controlling the reaction to some
extent by adjusting the amount of the catalyst used.
In the chemical polymerization method, however, since the gel reaction
takes place immediately after the addition of the polymerization initiator
to the monomer solution, giving rise to a viscosity increase of the
solution and to gelatinization of the mixture, it is necessary to prepare
a monomer solution and a polymerization initiator solution separately and
mix them just before the start of the gel forming process. This separate
addition of a peroxide solution and a reducing solution to the monomer
solution inevitably requires complicated steps. On the other hand, if
these two solutions are mixed together beforehand, the catalyst activity
will change as the time passes, making it difficult to stably obtain high
quality gels.
Furthermore, since a molecular sieve effect of a polyacrylamide gel varies
depending on the gel concentrations, various products with different gel
concentrations are required conforming to molecular weights or the like of
the components to be separated or analyzed. Conventionally, this
requirement for polyacrylamide gels has been fulfilled by preparing a
monomer solution having a specific gel concentration, which corresponds to
the subject to be separated for analysis, then adding a polymerization
initiator to the solution and introducing the mixture into or spreading it
over a gel-supporter. This necessitates to prepare many gel-forming
solutions with different concentrations when the subjects to be analyzed
contain a number of components. In addition, preparation of a gel with a
concentration gradient suitable for the analysis of a subject comprising a
wide molecular weight distribution is implemented by providing two monomer
solutions with different concentrations, one a low concentration and the
other a high concentration, and adding a polymerization initiator to the
solutions. The solutions are then introduced into or spread over a
gel-supporter by using a gradient forming unit. In order to obtain a
number of gels with different concentration gradients, it is necessary, in
the same manner as in the preparation of a series of gels with a specified
concentration, to prepare beforehand various monomer solutions with
different concentrations corresponding to the required gradients, and to
appropriately combine these solutions. Furthermore, in order to prepare a
stable gel, the amount of the polymerization initiator must be adjusted
appropriately so that not too less or not too much of it is incorporated
relative to the concentration of the monomer solution to be used.
As noted above, stable supply of high quality polyacrylamide gels in large
quantities has been difficult to achieve by conventional preparation
methods which involve rather complicated manufacturing steps and require
sophisticated skills.
An object of the present invention is, therefore, to provide an easy
production process which can prepare a large quantity of various kinds of
high quality and stable acqueous polyacrylamide gels, having a high
resolving power and any desired concentrations or concentration gradients,
with a good reproducibility, and which does not involve the problem of
changes in the polymerization initiator activity before being introduced
into a gel-supporter or does not require to prepare many monomer solutions
or many polymerization initiator solutions of different concentrations
beforehand.
In view of this situation, the present inventors have undertaken intensive
studies to solve the aforementioned problems, and found that a high
quality gel with a desired concentration can constantly and easily be
prepared with a good reproducibility and without any changes in the
catalyst activity using an optimum amount of a polymerization initiator,
by simply preparing one monomer solution with a high concentration, one
peroxide solution with a low concentration, and one reducing solution with
a low concentration, and mixing them at an appropriate ratio prior to use.
These findings have led to the completion of the present invention.
SUMMARY OF THE INVENTION
Accordingly, an object of this invention is to provide a process for
preparing a polyacrylamide gel plate for electrophoresis with a desired
concentration which comprises mixing an acrylamide monomer solution with a
high concentration, a peroxide solution with a low concentration, and a
reducing agent solution with a low concentration, at an arbitrary ratio,
and introducing the mixture into a gel-supporter.
In a preferred embodiment, the concentration of said acrylamide monomer
solution is in the range of 20-50% by weight, and the concentrations of
said peroxide solution and reducing agent solution are respectively
0.005-1.0% by weight; and said arbitrary concentration is in the range of
2-50 w/v %.
Another object of the present invention is to provide a process for
preparing said polyacrylamide gel plate for electrophoresis in which the
mixing of said acrylamide monomer solution, peroxide solution, and
reducing agent solution are implemented simultaneously, using or not using
a computer control system.
Still another object of the present invention is provide a process for
preparing said polyacrylamide gel plate for electrophoresis in which said
polyacrylamide gel has a plurality of concentrations or a concentration
gradient.
Other objects, features and advantages of the invention will hereinafter
become more readily apparent from the following description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the outline of a preferred embodiment of the process of the
present invention.
FIG. 2 shows an example of a mixing pattern of the three solutions of the
present invention; peroxide solution (A), reducing agent solution (B), and
high concentration monomer solution (C), wherein the concentrations are
changed stepwise.
FIG. 3 shows an example of a mixing pattern of the three solutions of the
present invention; peroxide solution (A), reducing agent solution (B), and
high concentration monomer solution (C), wherein the concentrations are
continuously changed.
FIG. 4 shows a liquid-transfer pattern of peroxide solution (A1), reducing
agent solution (A2), and high concentration monomer solution (A3) employed
in Example 2.
FIG. 5 shows a liquid-transfer pattern of peroxide solution (A1), reducing
solution (A2), and high concentration monomer solution (A3) employed in
Example 2.
FIG. 6 shows a liquid-transfer pattern of peroxide solution (A1), reducing
agent solution (A2), and high concentration monomer solution (A3) employed
in Example 2.
FIG. 7 shows an electrophoresis image of the gel plate obtained by the
liquid-transfer according to the pattern indicated in FIG. 4.
FIG. 8 shows an electrophoresis image of the gel plate obtained by the
liquid-transfer pattern indicated in FIG. 5.
FIG. 9 shows an electrophoresis image of the gel plate obtained by the
liquid-transfer according to the pattern indicated in FIG. 6.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
In the practice of the present invention, the solutions to be prepared
beforehand are only three kinds; one acrylamide monomer solution with a
high concentration, one peroxide solution with a low concentration, and
one reducing agent solution with a low concentration. There is no need to
prepare many kinds of monomer solutions beforehand. It is possible to
prepare a large amount of these solutions at a time and use it either
continually or recurrently whenever required by taking out a small amount
of these solutions.
The high concentration acrylamide monomer solution used in the present
invention contains an acrylamide monomer and a cross-linking agent, and as
required, an anionic surface active agent as a modifier, a pH buffering
agent, and the like.
Examples of acrylamide monomers include acrylamide homologues such as
acrylamide, N-methylacrylamide, N,N'-dimethylacrylamide,
N-(hydroxymethyl)acrylamide, diacetoneacrylamide, and the like;
methacrylamide; and the like. These compounds can be used individually or
in combination of two or more. Among these compounds, acrylamide is
preferred, and the combined use of acrylamide with one or more of other
acrylamide monomers is also possible.
As a cross-linking agent, any compounds which possess divalent, trivalent
or more cross-linking function can be used. Specific examples of the
divalent compounds are N,N'-methylenebisacrylamide (BIS),
N,N'-propylenebisacrylamide, diacrylamide dimethyl ether, piperazine
diacrylamide, and the like. Of these cross-linking agents, BIS is
preferred. One or more of these cross-linking agents can be used together
in combination.
As an anionic surface active agent, alkyl sulfates, particularly those
containing a long chain alkyl group with more than 10 carbon atoms are
preferable. The most preferred is sodium dodecyl sulfate (SDS).
Various types of pH buffering agents can be found by reference to
"Chemistry Handbook--Fundamental Part" (edited by The Chemical Society of
Japan) and the like. Enumerated as specific examples are
Tris(hydroxymethyl)aminomethane (Tris), N,N'-bis(2-hydroxyethyl)glycine,
sodium N-2-hydroxypiperazine-N'-2-hydroxypropane-3-sulfonate, as well as
any acids, alkalis, or salts used, as required, in combination with one of
these compounds. Tris-hydrochloride buffer (pH 7.0-9.2) is an example of
such a buffering agent which is most popularly used.
The concentration of the acrylamide monomer solution, in terms of the sum
of acrylamide monomers and cross-linking agents contained, used in the
present invention is preferably higher than the concentration usually
required, for example, 10-98% by weight, and more preferably 20-50% by
weight. The amount of the cross-linking agent is preferably in the range
of 1-30% by weight, with the particularly preferable range being 2-10% by
weight, based on the total amount of the monomer and the cross-linking
agent.
Preparation of the acrylamide monomer solution may be implemented by
dissolving or dispersing the aforementioned components into water or a
mixed solution of water and an organic solvent.
The peroxide compound to be used in the present invention may be any
peroxides disclosed in known references on electrophoresis
("Electrophoresis--Fundamentals and Experiments", edited by H. Terada, and
the like.) and includes ammonium peroxodisulfate, an alkali metal
peroxodisulfate, and the like.
The concentration of the peroxide solution must be low, for example,
0.001-5.0% by weight, or more preferably 0.005-1.0% by weight, from the
aspect of its functions as a diluent for the monomer solution and as a
polymerization initiator. Preparation of the peroxide solution may be
implemented by dissolving or dispersing said peroxide and, as required,
anionic surface active agents, pH buffering agents, and the like into
water or a mixed solution of water and an organic solvent.
The reducing agents to be used in the present invention may be amine
compounds disclosed in known references on electrophoresis
("Electrophoresis--Fundamentals and Experiments", edited by H. Terada, and
the like.). Specific examples include N,N,N',N'-tetramethylethylenediamine
(TEMED), N,N,-dimethylethylenediamine, 3-dimethylamino-n-propylamine,
3-dimethylaminopropionitrile, N-n-butyldimethylamine,
N,N'-dimethylpiperazine, and the like.
The concentration of the reducing agent solution must be low, for example,
0.001-5.0% by weight, and more preferably 0.005-1.0% by weight, from the
aspect of its functions as a diluent for the monomer solution and as a
polymerization initiator. Preparation of the reducing agent solution may
be implemented by dissolving or dispersing said reducing agent and, as
required, anionic surface active agents, pH buffering agents, and the like
into water or a mixed solution of water and an organic solvent.
When preparing a mixed solution of any desired gel concentration by mixing
these three solutions, namely a high concentration acrylamide monomer
solution, a low concentration peroxide solution, and a low concentration
reducing solution, which have been separately prepared beforehand, it is
desirable to mix and stir these three solutions at a time in order to
avoid the occurrence of reactions between peroxides and reducing agents.
The term "any desired gel concentration" herein means that the
concentration of the acrylamide monomer and the crosslinking agent
contained in the gel can arbitrarily be controlled. Usually, the gel
concentration is adjusted in the range of 2-50 w/v %. The introduction of
the mixed solution to a gel-supporter can be performed by transferring the
solution or spreading it over the gel-supporter. As the gel-supporter, any
known support material which is conventionally used, such as a glass tube,
a glass plate, an organic polymer, or the like, can be employed.
A preferable example of the process of mixing these three solutions and
introducing it into the gel-supporter is presented in FIG. 1. The three
solutions, peroxide solution (A), reducing agent solution (B), and monomer
solution (C), are transferred respectively by pumps (P), and stirred and
mixed by a mixer (M) and introduced into the gel-supporter. In this
process, any target solutions having a desired gel concentration can be
prepared by adjusting the flow ratio of the pumps by which the three
solutions are transferred. In addition, if the flow ratio of the pumps is
designed to be controlled by a computer (PC) via a controller (CB), the
gel concentrations of the solution can be changed stepwise as shown in
FIG. 2, or it is possible to form a concentration gradient easily by
changing the flow ratio continuously as shown in FIG. 3.
The electrophoresis gel plates prepared by this invention are desired to be
substantially colorless and transparent for making it easy to detect and
read the electrophoresis images.
The electrophoresis gel plates prepared by this invention can be applied to
horizontal or vertical electrophoresis, and the like, according to a known
method disclosed in the aforementioned literature, patent applications,
and the like.
Other features of the invention will become apparent in the course of the
following description of the exemplary embodiments which are given for
illustration of the invention and are not intended to be limiting thereof.
EXAMPLES
Example 1
Gel-forming Solutions A (A1-A3) of the present invention and Comparative
Solutions B, consisting of Solution (B1) which is a mixture of a peroxide
and a reducing agent and Solution (B2) which is a mixture of acrylamide
and a cross-linking agent, were prepared using the formulations given in
Table 1. These solutions were degassed to adjust the content of dissolved
oxygen and stored at 4.degree. C. under a helium atmosphere. After certain
periods of time specified in Table 2, each solution was taken out and
mixed together at 4.degree. C. with a specified ratio to obtain solutions
of the same gel-content. After mixing, the time required for each solution
to begin to gelatinize, i.e, the period of time required for the
temperature of the solution to begin to rise by the exothermic reaction
(hereinafter referred to as "Gelatinized Time"), was measured and
determined as given in Table 2.
The Gelatinized Time for Solution A showed a constant value irrespective of
the time elapsed, whereas that for Solutions B varied depending on the
elapsed periods, and ultimately, Solution B showed no gelatinization after
24 hours, even though Solutions B1 and B2 were mixed. In addition, when
slab-type electrophoresis gel plates were prepared from Solution A and
Solution B, which had been prepared and stored under the same conditions,
the gel plates prepared from Solution A did not show any effects due to
the elapsed time, while the gel plates from Solution B gave a stringy
appearance as a whole as the time elapsed, producing strains on phoresis
images.
TABLE 1
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Components for
Invention Composition A
Gel Forming Peroxide Solution
Reducing Agent
Monomer Solution
Comparative Composition B
Solution (A1) Solution (A2)
(A3) B1 B2
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Acrylamide -- -- 390
g -- 390
g
BIS -- -- 10 g -- 10 g
pH buffering agent
250
ml 250
ml 250
ml 250
ml 250
ml
APS 0.07
g -- -- 0.035
g --
TEMED -- 0.06
ml 0.03
ml 0.03
ml 0.03
ml
Water Balance Balance Balance Balance
Balance
(an amount making the
total volume 1000 ml)
__________________________________________________________________________
Note:
BIS: N,Nmethylenebisacrylamide (Crosslinking agent)
pH buffer: 1.5M Tris(hydroxymethyl)aminomethanehydrocholide, pH 8.8
APS: Ammoniumperoxodisulfate (Peroxide)
TEMED: N,N,N',Ntetramethylethylenediamine (Reducing agent)
TABLE 2
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Gelatinized
Time Elapsed Time (hours)
(minutes) 0 1 4 8 12 24
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Solution 35 34 35 33 34 35
Solution 35 30 30 45 80 --*
B
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Note: "Solution A" was formulated with a mixing ratio of A1:A2:A3 = 3:3:2
while "Solution B" was formulated with a mixing ratio of B1:B2 = 3:1, at
every elapsed time, to prepare gelforming solutions containing 10%
acrylamide.
*Solution B was not gelatinized at 24 hours or later.
As illustrated above, the process of this invention can avoid changes in
the catalyst activity due to interactions between a peroxide and a
reducing agent so that the target gels can always be produced under
consistent polymerization conditions, with a good reproducibility.
Furthermore, the solutions once prepared can be used continually or kept
stored over a long period of time.
Example 2
Gel-forming Solutions A (A1-A3) of the present invention were prepared
according to the formulations given in Table 1, and the solutions were
served for producing slab-type electrophoresis gel plates using an
equipment shown in FIG. 1 by changing the flow ratio as shown in FIGS.
4-6. The gel plates thus prepared were subjected to SDS-polyacrylamide gel
electrophoresis according to a known reference on electrophoresis using a
mixture of proteins whose molecular weights were known, as a
molecular-weight marker. The electrophoresis images obtained in this test
are presented in FIGS. 7-9. According to the process of this invention,
any gels with a desired gel concentration or a desired concentration
gradient can be produced by simply changing the mixing pattern of three
solutions. This versatility is quite advantageous for preparing many kinds
of products without complicated procedure changes.
High quality polyacrylamide gel plates for electrophoresis, which are
useful for analysis of high molecular weight in vivo components typified
by proteins, can be prepared in large quantities with a good
reproducibility by the process of this invention. This is not only
advantageous for the production of various gels with different gel
concentrations or different concentration gradients, but also serves for
improving the productivity of multi-products manufacturing.
Obviously., numerous modifications and variations of the present invention
are possible in light of the above teachings. It is therefore to be
understood that within the scope of the appended claims, the invention may
be practiced other than as specifically described herein.
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