JP2003279539A - Electrophoresis method optimized to grain protein, gel for electrophoresis used therefor, buffer solution, and sample extract - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To achieve a clear migration pattern in a shorter time than by a conventional method when electrophoresis is performed on grain proteins. <P>SOLUTION: Eelectrophoresis is performed on the grain protein with three essential elements of (1) a polyacrylamide gel of which the maximum degree of swelling in water is between 1.3-2.0, (2) a sample extract for SDS-PAGE containing of a thiolic reducer which corresponds to a large excess amount to the number of cysteine residual groups in the sample proteins in a buffer solution of pH 7.0-9.0, and (3) a migration buffer solution both having negative charge while holding SH groups in a migration buffer solution (tris (hydroxymethyl) aminomethane of 25 mM, glycine of 192 mM, (w/v) SDS of 0.1%) of Laemmli method composition, a discontinuous buffer solution of SDS- PAGE, and containing the thiolic reducer to be developed into the gel during electrophoresis. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).
polyacramide gel electr
ophoresis method), and particularly to analytical tools that contribute to the study of grain proteins, which has heretofore been difficult to separate and analyze.

[0002]

2. Description of the Related Art The conventional method for separating and analyzing proteins by SDS-PAGE (Laemmli et al.
mliU. K. : 1970, Nature, 227, 6
80-685) has been used exclusively. Attempts have been made to separate various proteins using this method, but some proteins have the drawback of being difficult to separate. For example, when grain proteins such as wheat were attempted to be separated by the method of Laemmli et al., The difference between the protein bands was small and separation was difficult. (See Figure 2). Therefore, Bushuk
Modified method (Bushuk, Cereal Chem
Istry, 64 (4), 324-327) was proposed, but the operation takes time as shown in the following "Comparison table of operation time by the conventional method (Bushuk method) and the method of the present invention" (Table 1). . Further, as shown in comparison with FIGS. 4 and 5, there is a drawback that the migration pattern is blurred.

[0003]

DISCLOSURE OF THE INVENTION The present invention is directed to an electrophoretic method capable of obtaining a clear electrophoretic pattern in a shorter time than the conventional method (Laemmli method or Bushuk method) in this kind of technique, and a gel and an electrophoretic buffer used therefor. , Providing a sample extract.

[0004]

First, in carrying out the method of the present invention, the following three components are essential. The first is a polyacrylamide gel having a maximum degree of swelling in water of 1.3 to 2.0. Here, the maximum swelling degree is the maximum swelling degree = the gel volume after the water swelling after polymerization / the volume of the gel solution before the polymerization, and the maximum swelling degree in water is constant regardless of the gel concentration (% T). , Varying only to the degree of cross-linking (% C) in polyacrylamide gels. The relationship is shown in FIG. This graph shows the average value and standard error of the maximum swelling degree for each gel concentration (% T). In SDS-PAGE, proteins are developed in a polyacrylamide gel polymerized in an aqueous solution, while the maximum swelling degree in water of the polyacrylamide gel used in the method of Laemmli et al. It lacks flexibility as a carrier for separating proteins. That is, as the first component, the maximum swelling degree in water is 1.1 or more, particularly 1.3 to
It is optimal for separating grain proteins that the polyacrylamide gel of 2.0 has a flexible structure as a separation carrier. (See Figures 1 and 2)

The following constituents are SDS containing a thiol reducing agent corresponding to a large excess amount with respect to the number of cysteine residues in the sample protein in a buffer solution having a pH of 7.0 to 9.0 or less.
-A sample extract for PAGE. Since it is difficult to extract wheat protein with a sample extract commonly used in SDS-PAGE including the method of Bushuk et al.,
After addition of the sample extract, there was no choice but to stir for a long time (2 hours) for extraction. It is generally known that wheat flour forms an insoluble precipitate called gluten when it is kneaded with water. This is because the proteins in wheat, glutenin and gliadin, are strongly bound via the SH group of cysteine in the protein. This is to form a macromolecule. On the other hand, the above bond in the protein is called SS bond and pH
It has been found that the reactivity is most enhanced at 7.0 to pH 9.0 and is easily reduced to the original cysteine. Since the pH of the conventional sample extract is 6.8, the reactivity of the thiol reducing agent in the sample extract is low and it takes a long time to extract wheat protein. Therefore, the most reactive pH 7.0
It was found that a sample extract containing a large excess amount of a thiol reducing agent in a buffer solution of up to 9.0 can be extracted with an extremely short stirring time. (See Figure 3)

As the third component, SDS-PAGE
Laemmli method composition running buffer (25 mM tris (hydroxymethyl) aminomethane, 192 mM glycine, 0.1% (w / v) SDS)
It is a migration buffer containing a thiol reducing agent that has a negative charge while retaining the SH group and is developed into a gel during electrophoresis. When kneading bread dough from wheat flour to make bread, macromolecules are formed by repeatedly breaking and re-binding the wheat protein glutenin and gliadin cysteine via the SH group. I know that On the other hand, the same phenomenon in principle
It can occur during DS-PAGE, that is, proteins separated during electrophoresis repeat SS bond cleavage and recombination, and thus the method of Bushuk et al. Was not reproducible in the results and the migration pattern was said to be blurred. . Therefore, S
If the H group is constantly supplied into the gel during SDS-PAGE, the above-mentioned action is suppressed, that is, the SH group is maintained in the discontinuous buffer solution of SDS-PAGE, and it has a negative charge and can be developed into the gel with a thiol property. It was found that the migration pattern became clear by using the migration buffer containing the reducing agent.

FIG. 4 shows a comparison of migration patterns between the case where the conventional buffer solution is used () and the case where the buffer solution is used (). Especially, the blurring in () is extremely clear in () figure. Is appearing. When SDS-PAGE electrophoresis is performed using the above three constituents (SDS-PAGE of wheat protein), a clear migration pattern can be obtained in a short time.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The most preferred embodiment of the present invention will be described below. First, the maximum degree of swelling in water is 1.3-2.
About making a polyacrylamide gel that becomes 0. 1 m between two flat glass plates (120 x 102 x 3 mm)
Prepare 2 sets of assembled glass plates with a space of m and sealing the bottom surface to prevent water from leaking. Two types of separation gel solutions having the compositions shown in the following table "Comparison table of gel compositions in the conventional method (Laemmli method) and the method of the present invention" (Table 2) were prepared, and the position 1.5 cm from the upper end of the above glass plate. Pour up to. Water is gently overlaid and left at room temperature for about 60 minutes.
After polymerization of the separated gel, the top end of the separated gel is washed with a small amount of two kinds of concentrated gel solutions having the compositions shown in Table 2 prepared in advance, and the remaining concentrated gel solution is poured to reach the upper end of the glass plate. Insert a comb (made of plastic and toothed) and leave at room temperature for about 30 minutes. Remove comb and sealant. After immersing the two gels in water and swelling the gel, the maximum swelling degree was measured. After preparing a gel of the same composition again as described above, this glass plate was set in an electrophoresis tank, and a running buffer solution (25 m) having a Laemmli method composition was prepared.
M tris (hydroxymethyl) aminomethane, 192m
M glycine, 0.1% (w / v) SDS) was poured, and the wheat sample that had undergone sample extraction (see chart below) was added to the concentrated gel sample hole, the migration tank was connected to the migration power supply device, and the constant current was 20 mA. Electrophoresis was performed for about 75 minutes. "Detailed chart of wheat sample extraction conditions" Wheat 40 mg ↓ + 70% (w / v) ethanol 1 mL ↓ Agitation (room temperature, 60 minutes) ↓ Centrifuge (12000 g, room temperature, 20 minutes) Precipitation → Discard supernatant ↓ + Sample Extraction liquid 1mL ↓ Stir (room temperature, few seconds) ↓ Boiling (100 ° C hot water, 2.5 minutes) ↓ Centrifuge (12000g, 4 ° C, 20 minutes) Discard the precipitate → Use 0.5mL of supernatant as the analytical sample Remove the gel after electrophoresis from the glass plate and
% (V / v) methanol, 7.5% (v / v) acetic acid) and shake for 30 minutes at room temperature to immobilize the protein in the gel. The fixative is discarded, and the CBB staining solution (50% (v /
v) methanol, 10% (v / v) acetic acid, 0.1% (w
/ V) CBB (Coomassie Brilliant Blue R25
0)), heat in a microwave oven for 30 seconds, and shake at room temperature for about 30 minutes to stain the protein in the gel with a dye. Discard the CBB staining solution and remove the decolorizing solution (30% (v / v)
Immerse in methanol, 10% (v / v) acetic acid), heat in a microwave oven for 30 seconds, and shake at room temperature for about 30 minutes to wash and decolor the dye other than the protein portion. Repeat the bleaching operation until you can clearly see the protein band. Take out from the decolorizing solution and save the migration pattern as image data using an image analyzer or digital camera. By the above operation, as shown in FIG. 2, in the gel having the maximum swelling degree of 1.9, there was a large difference in the mobility of each protein band, and good separation could be performed. On the other hand, in the gel having the maximum swelling degree of 1.1, the difference in mobility between protein bands was small and separation was difficult.

Next, SH groups were added to a running buffer (25 mM tris (hydroxymethyl) aminomethane, 192 mM glycine, 0.1% (w / v) SDS) having a Laemmli method composition, which is a discontinuous SDS-PAGE buffer. A running buffer containing a thiol-reducing agent that has a negative charge and is developed into a gel during electrophoresis. Two pairs of flat glass plates (120 × 102 × 3 mm) having a hollow of 1 mm between them and having their bottom surfaces sealed to prevent water from leaking are prepared. The above-mentioned conventional method (Lae
mmli method) and the gel composition comparison method of the present invention (Table 2), two separate gel solutions having the compositions of the present invention are prepared and poured to a position of 1.5 cm from the upper end of the glass plate. Gently overlay with water and leave at room temperature for about 60 minutes. After polymerization of the separated gel, the top end of the separated gel is washed with a small amount of two kinds of concentrated gel solutions having the compositions shown in Table 2 prepared in advance, and the remaining concentrated gel solution is poured to reach the upper end of the glass plate. Insert the comb (made of plastic and toothed) and leave at room temperature for about 30 minutes. Remove comb and sealant. When the maximum degree of swelling was measured after swelling by immersing two gels in water, the gel of the present invention showed 1.9. After preparing the gel of the same composition again as described above, each glass plate was set in the electrophoresis tank, and one was a running buffer (25 m) of the Laemmli method composition.
M tris (hydroxymethyl) aminomethane, 192m
M glycine, 0.1% SDS), and the other is the running buffer of the present invention (25 mM tris (hydroxymethyl) aminomethane, 192 mM glycine, 0.1% (w
/ V) SDS, 50 mM L-Cysteine) is poured, a wheat sample that has undergone sample extraction (see the chart above) is added to the pores of the concentrated gel sample, the migration tank is connected to a migration power supply, and a constant current of about 20 mA is applied for about 75 Electrophoresed for minutes. Remove the gel after electrophoresis from the glass plate and fix it (20%
Immobilize the protein in the gel by soaking in (v / v) methanol, 7.5% (v / v) acetic acid) and shaking for 30 minutes at room temperature. The fixative is discarded, and the CBB staining solution (50% (v /
v) methanol, 10% (v / v) acetic acid 0.1% (w /
v) CBB (Coomassie Brilliant Blue R250))
Soak in a microwave oven for 30 seconds, then at room temperature for about 30 seconds
Shake for minutes to stain the proteins in the gel. CB
Discard the B staining solution, immerse in the decolorization solution (30% (v / v) methanol, 10% (v / v) acetic acid), and use a microwave oven for 3
After heating for 0 second, shake at room temperature for about 30 minutes to wash the pigments other than the protein portion to decolorize. Repeat the bleaching operation until you can clearly see the protein band. Take out from the decolorizing solution and save the migration pattern as image data using an image analyzer or digital camera. As a result of the above operation, a clear electrophoretic pattern was obtained with this buffer solution, as shown in FIG. On the other hand, as shown in FIG.
The migration pattern was blurred with the buffer solution in the li method.

Further, a sample treatment solution for SDS-PAGE containing a thiol reducing agent corresponding to a large excess amount with respect to the number of SH groups in the sample protein in a buffer solution having a pH of 7.0 to 9.0 or less is prepared. Will be described. Flat glass plate (12
0x102x3mm) with a 1mm hollow between the two,
Prepare one set of assembled glass plates with their bottom surfaces sealed to prevent water from leaking. The above-mentioned conventional method (Laemml
Method i) and the method of the present invention, a separating gel solution having the composition of the present invention shown in the comparison table is prepared and poured into a position of 1.5 cm from the upper end of the glass plate. Water is gently overlaid and left at room temperature for about 60 minutes. After polymerization of the separated gel, the top end of the separated gel is washed with a small amount of the concentrated gel solution having the composition shown in the above-mentioned comparative table, and the remaining concentrated gel solution is poured to reach the upper end of the glass plate. Insert a comb (made of plastic and toothed) and leave at room temperature for about 30 minutes. Remove comb and sealant. The gel of the present invention showed 1.9 when the maximum swelling degree was measured after swelling by immersing the gel in water. After preparing a gel of the same composition again as described above, the glass plate was set in the electrophoresis tank, and the running buffer solution of the Laemmli method composition (25 mM tris (hydroxymethyl) aminomethane, 192 mM glycine, 0.1% (w / V) SDS)
Pour. Based on the sample extraction method shown in the above chart, 40 mg × 18 samples of wheat samples were mixed with 1 ml of 70% ethanol, and 6 samples each were stirred for 0, 10, 60 minutes at room temperature. Each of them was centrifuged and the supernatant was discarded, and the precipitates of the Laemmli method and the sample extract of the present invention having the composition shown in the following “Comparative table of sample extract composition of the conventional method and the present invention” (Table 3) 1 ml was added, and the mixture was stirred at room temperature for 0, 10, 60 minutes. It was boiled in boiling water for 2.5 minutes, centrifuged at 4 ° C. for 20 minutes, and the supernatant was collected to give a sample for electrophoresis. This sample was added to the pores of the concentrated gel sample, the migration tank was connected to a migration power supply device, and electrophoresis was performed at a constant current of 20 mA for about 75 minutes. The gel after electrophoresis was removed from the glass plate, and the fixing solution (20% (v / v) methanol, 7.5% (v / v)
v) Soak in acetic acid) and shake for 30 minutes at room temperature to fix the protein in the gel. After discarding the fixative solution, soak in CBB staining solution (50% (v / v) methanol, 10% (v / v) acetic acid 0.1% (w / v) CBB (Coomassie Brilliant Blue R250)) and microwave. After heating for 30 seconds, shake at room temperature for about 30 minutes to stain the protein in the gel with a dye. Discard the CBB staining solution and remove the decolorization solution (30%
Dip in (v / v) methanol, 10% (v / v) vinegar,
After heating in a microwave oven for 30 seconds, shake at room temperature for about 30 minutes,
Dyes other than protein are washed away. Repeat the bleaching operation until you can clearly see the protein band.
Take out from the decolorizing solution and save the migration pattern as image data using an image analyzer or digital camera.
As shown in FIG. 3, the density of common protein bands in each lane (hereinafter Density) was compared. As a result, the maximum value 105 of the conventional method was obtained by stirring with 70% ethanol for 60 minutes and with the sample extract of the present invention for 0 minutes. That is, it was found that the conventional sample treatment liquid could not be extracted unless it was stirred for 1 hour, but in the present invention, an equivalent amount was extracted by stirring for an extremely short time.

The above is the embodiment of the present invention. Three examples of the present invention will be shown below.

[0012]

[Example 1] In the case where the wheat varieties produced in 1997 are discriminated by the SDS-PAGE method of the present invention. 1. Preparation of Gel Prepared is a glass plate (120 × 102 × 3 mm) having two 1 mm-thick hollows and a bottom sealed so that water does not leak. A separating gel solution having the composition of the present invention shown in Table 2 is prepared and poured to a position 1.5 cm from the upper end of the glass plate. Layer the water gently,
Leave at room temperature for about 60 minutes. After the separation gel polymerization, the top end of the separation gel is washed with a small amount of the concentrated gel solution having the composition shown in Table 2 which has been prepared in advance, and the remaining concentrated gel solution is poured to reach the top end of the glass plate. Insert a comb (made of plastic and toothed) and leave at room temperature for about 30 minutes. Remove comb and sealant. The gel of the present invention showed 1.9 when the maximum swelling degree was measured after swelling by immersing the gel in water.

2. Sample preparation 1 ml of 70% ethanol was added to 40 mg of flour of each variety and stirred at room temperature for 60 minutes. After centrifugation and discarding the supernatant, 1 ml of the sample extract of the present invention having the composition shown in Table 3 was added to this precipitate, and the mixture was stirred at room temperature for several seconds. It was boiled in boiling water for 2.5 minutes, centrifuged at 4 ° C. for 20 minutes, and the supernatant was collected to prepare a sample for electrophoresis.

3. The electrophoresis glass plate was set in the electrophoresis tank, and the electrophoresis buffer solution of the present invention 25 mM tris (hydroxymethyl) aminomethane, 1
92 mM glycine, 0.1% (w / v) SDS, 50 m
(ML-Cysteine), the wheat sample that has undergone sample extraction (see the chart above) is added to the concentrated gel sample hole, the migration tank is connected to the migration power supply device, and the constant current is 20 mA.
Electrophoresis was performed for about 75 minutes. Remove the gel after electrophoresis from the glass plate, and fixative (20% (v / v) methanol,
Immobilize the protein in the gel by soaking in 7.5% (v / v) acetic acid and shaking for 30 minutes at room temperature. Discard the fixative,
CBB stain (50% (v / v) methanol, 10%
(V / v) Acetic acid 0.1% (w / v) CBB (Coomassie Brilliant Blue R250)), heated in a microwave oven for 30 seconds, and then shaken at room temperature for about 30 minutes to stain the protein in the gel as a dye. To dye. Discard the CBB staining solution, soak it in the decolorizing solution (30% (v / v) methanol, 10% (v / v) acetic acid), heat it in the microwave for 30 seconds, and then at room temperature for about 3
Shake for 0 minutes to wash and decolor the dye except the protein portion. Repeat the bleaching operation until you can clearly see the protein band. Take out from the decolorizing solution and save the migration pattern as image data using an image analyzer or digital camera.

4. Results It was found that the present invention enables discrimination between 1997 domestic wheat varieties. (Fig. 5)

[0016]

[Example 2] In the case of determining the variety of domestic wheat produced in 2001 by the SDS-PAGE method in the present invention. 1. Preparation of Gel Prepared is a glass plate (120 × 102 × 3 mm) having two 1 mm-thick hollows and a bottom sealed so that water does not leak. A separating gel solution having the composition of the present invention shown in Table 2 is prepared and poured to a position 1.5 cm from the upper end of the glass plate. Layer the water gently,
Leave at room temperature for about 60 minutes. After the separation gel polymerization, the top end of the separation gel is washed with a small amount of the concentrated gel solution having the composition shown in Table 2 which has been prepared in advance, and the remaining concentrated gel solution is poured to reach the top end of the glass plate. Insert the comb (made of plastic and toothed) and leave at room temperature for about 30 minutes. Remove comb and sealant. The gel of the present invention showed 1.9 when the maximum swelling degree was measured after swelling by immersing the gel in water.

2. Sample preparation 1% of 70% ethanol was added to 40 mg of flour of each variety and stirred for 60 minutes at room temperature. Centrifuge and discard the supernatant,
1 ml of the sample extract of the present invention having the composition shown in Table 3 was added to the precipitate, and the mixture was stirred at room temperature for several seconds. It was boiled in boiling water for 2.5 minutes, centrifuged at 4 ° C. for 20 minutes, and the supernatant was collected to give a sample for electrophoresis.

3. The electrophoresis glass plate was set in the electrophoresis tank, and the electrophoresis buffer solution of the present invention (25 mM tris (hydroxymethyl) aminomethane,
192 mM glycine, 0.1% (w / v) SDS, 50
(mM L-Cysteine), the wheat sample that has been sample-extracted (see the chart above) was added to the concentrated gel sample hole, the migration tank was connected to the migration power supply device, and the constant current was 20 m.
Electrophoresis was performed at A for about 75 minutes. Remove the gel after electrophoresis from the glass plate, immerse it in a fixative solution (20% (v / v) methanol, 7.5% (v / v) acetic acid) and shake it at room temperature for 30 minutes to remove the protein in the gel. Fix it. After discarding the fixative, a CBB stain (50% (v / v) methanol, 10
% (V / v) acetic acid, 0.1% (w / v) CBB (Coomassie Brilliant Blue R250)), heat in microwave oven for 30 seconds, shake at room temperature for about 30 minutes, and then remove the protein in the gel. To dye. The CBB staining solution is discarded and the decolorization solution (30% (v / v) methanol, 10% (v / v)
After being soaked in acetic acid) and heated in a microwave oven for 30 seconds, it is shaken at room temperature for about 30 minutes to wash and decolor the dye other than the protein portion. Repeat the bleaching operation until the protein band is sufficiently visible. Take out from the decolorizing solution and save the migration pattern as image data using an image analyzer or digital camera.

4. Results According to the present invention, it was found that the domestic wheat varieties produced in 2001 can be distinguished from each other (see FIG. 6). It was also found that there was no difference in the pattern before and after milling.

[0020]

[Example 3] In the case of SDS-PAGE according to the present invention of wheat of the same variety having different production areas and production years. 1. Preparation of Gel Prepared is a glass plate (120 × 102 × 3 mm) having two 1 mm-thick hollows and a bottom sealed so that water does not leak. A separating gel solution having the composition of the present invention shown in Table 2 is prepared and poured to a position 1.5 cm from the upper end of the glass plate. Layer the water gently,
Leave at room temperature for about 60 minutes. After the separation gel polymerization, the top end of the separation gel is washed with a small amount of the concentrated gel solution having the composition shown in Table 2 which has been prepared in advance, and the remaining concentrated gel solution is poured to reach the top end of the glass plate. Insert the comb (made of plastic and toothed) and leave at room temperature for about 30 minutes. Remove comb and sealant. The gel of the present invention showed 1.9 when the maximum swelling degree was measured after swelling by immersing the gel in water.

2. Sample preparation 1 ml of 70% ethanol was added to 40 mg of flour of each variety and stirred at room temperature for 60 minutes. Centrifuge and discard the supernatant, and in the precipitate 1 ml of the sample extract of the present invention having the composition shown in Table 3
Was added and stirred at room temperature for several seconds. It was boiled in boiling water for 2.5 minutes, centrifuged at 4 ° C. for 20 minutes, and the supernatant was collected to give a sample for electrophoresis.

3. The electrophoresis glass plate was set in the electrophoresis tank, and the electrophoresis buffer (25 mM tris (hydroxymethyl) aminomethane 1 of the present invention was used.
92 mM glycine, 0.1% (w / v) SDS, 50 m
ML-Cysteine) was poured, a wheat sample that had undergone sample extraction (see the chart above) was added to the pores of the concentrated gel sample, the electrophoresis tank was connected to the electrophoresis power supply device, and electrophoresis was performed at a constant current of 2 mA for about 75 minutes. Remove the gel after electrophoresis from the glass plate, and fixative (20% (v / v) methanol,
Immobilize the protein in the gel by soaking in 7.5% (v / v) acetic acid and shaking for 30 minutes at room temperature. Discard the fixative,
CBB stain (50% (v / v) methanol, 10%
(V / v) Acetic acid 0.1% (w / v) CBB (Coomassie Brilliant Blue R250)), heated in a microwave oven for 30 seconds, and then shaken at room temperature for about 30 minutes to stain the protein in the gel as a dye. To dye. Discard the CBB staining solution, immerse in the decolorization solution (30% (v / v) methanol, 10% (v / v) acetic acid), heat for 30 seconds in a microwave oven, shake at room temperature for about 30 minutes, and Dye off the area except the part. Repeat the bleaching operation until the protein band is sufficiently visible. Take out from the decolorizing solution and save the migration pattern as image data using an image analyzer or digital camera.

4. Results According to the present invention, it was found that wheat flour preserves the characteristics of varieties well even if the production area and year are different. (See Figure 7)

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the degree of crosslinking of polyacrylamide gel and the maximum degree of swelling in pure water.

FIG. 2 is a view showing an example of respective patterns for comparing the results of electrophoresis separation () of the grain protein by SDS-PAGE according to the conventional method and the results of electrophoresis separation according to the present invention ().

FIG. 3 is a diagram showing an example of migration patterns () when a buffer solution is used in a conventional method and migration patterns when a buffer solution is used in the method of the present invention) for comparing sample processing times. The conditions of each lane in these patterns are shown in ().

FIG. 4 is a diagram showing actual examples of migration patterns for comparing the conventional method () and the method of the present invention ().

FIG. 5: Migration pattern () and condition of each lane () showing the result of cultivar discrimination according to the present invention for domestic wheat in 1997
FIG.

FIG. 6: Migration pattern () showing conditions of 2001 domestic wheat cultivar discrimination according to the present invention and conditions of each lane ()
FIG.

FIG. 7 is a diagram showing migration patterns () showing the results of SDS-PAGE according to the present invention for wheat of the same variety having different production regions and production years, and conditions () of each lane.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 27/26 325B

Claims (4)

[Claims] 1. The maximum swelling degree in water is 1.3 to 2.0.
Sample for SDS-PAGE, which contains a polyacrylamide gel that becomes: 2) a thiol reducing agent corresponding to a large excess amount with respect to the number of cysteine residues in the sample protein, in a buffer solution having a pH of 7.0 to 9.0 or less Extract and 3) Laemm, a discontinuous buffer for SDS-PAGE
Running buffer solution of li method (25 mM tris (hydroxymethyl) aminomethane, 192 mM glycine, 0.1%
(W / v) SDS) and a thiol reducing agent that holds a thiol group (hereinafter SH group) and has a negative charge and is developed into a gel during electrophoresis. Electrophoresis method optimized for grain proteins, which is characterized. 2. A polyacrylamide gel used in the method according to claim 1, which has a maximum swelling degree in water of 1.3 to 2.0. 3. A pH used for carrying out the method according to claim 1.
A sample extract for SDS-PAGE, which comprises a thiol reducing agent corresponding to a large excess amount with respect to the number of cysteine residues in the sample protein in a buffer solution of 7.0 to 9.0 or less. 4. A discontinuous buffer solution for SDS-PAGE, L
running buffer (25 mM tris (hydroxymethyl) aminomethane, 192 mM glycine,
The method according to claim 1, further comprising a thiol reducing agent having a negative charge while retaining SH groups at 0.1% (w / v) SDS and developed into a gel during electrophoresis. Running buffer used for.