KR20020068818A - Polyacrylamide single gel for the use protein electrophoresis - Google Patents

Abstract

PURPOSE: Provided is a polyacrylamide single gel for a protein electrophoresis, which can stack protein with high resolution by stacking the protein on a gel loading buffer without a stacking gel. CONSTITUTION: The polyacrylamide single gel is a water solution containing an amine and a positive electrolyte in the molar ratio of 1:0.01-0.3, wherein the concentration of the amine is 0.3-1.0mol/l and the pH of the electrolyte solution is 7.5-11. And the amine is tris(hydroxy methyl)amino methane and the positive electrolyte is glycine, serine, asparagine, histidine, alanine, phenyl alanine, glutamine, tryptophan, glutamic acid, and etc. or derivatives thereof.

Description

Polyacrylamide single gel for the use protein electrophoresis

The present invention relates to a polyacrylamide monogel for electrophoresis widely used for protein separation in the biological sciences, such as biology, medicine, biochemistry.

Gel electrophoresis method using a polyacrylamide as a support can control the size of the micropath of the gel by the concentration and ratio of acrylamide and crosslinking agent to separate biomolecules of various sizes, and to provide protein for a long time due to high precision and high resolution. , Nucleic acids and the like have been widely used. In addition, when the polyacrylamide gel is used, the protein can be separated according to the intrinsic charge of the protein, and the protein can be separated according to the intrinsic weight. .

Protein electrophoresis using acrylamide as a support can be divided into two types, a continuous gel system using only one gel and a discontinuous buffer method using two gels.

The continuous gel system, that is, the method of separating proteins using only one gel, is a method of electrophoresis by selecting one buffer solution between pH 3 and 11 to make the same composition and concentration of electrolyte in the gel and the negative electrode solution. The continuous gel system is simpler to prepare than the discontinuous method and has less problems such as protein precipitation and aggregate formation in the sample, but has the disadvantage of not being able to electrophoresis a large volume of the sample. Also in terms of resolution, it is considerably lower than the discontinuous gel system and is rarely used at present.

In order to solve the above problems, Orstein and Davies discontinuous gel system to increase the resolution of the protein in the separation gel by first passing the stacking gel (stacking gel) before the protein sample is separated from the separating gel (separating gel) Ornstein, L. (1964) Ann. NY Acad. Sci., 121, 321; Davis, B.J. (1964) Ann. NY Acad. Sci., 121, 404].

Dodecylsilicate-discontinuous gel electrophoresis, the most widely used electrophoresis method for protein separation, was developed by Lamley [Laemmli, U.K. (1970) Nature 227, 680. Subsequently, in several papers and literatures, this method was slightly modified to introduce protein electrophoresis methods for specific situations, but the use of discontinuous gels, that is, basic gels and separation gels, which are the basic techniques, did not change.

The separation gel and the accumulation gel have similar chemical composition (tris (hydroxymethyl) aminomethane buffer solution, acrylamide / N, N-bisacrylamide, dodecyl lactate, etc.), but each compounding ratio and concentration of acrylamide, pH has different characteristics.

Accumulation gel serves to transfer the ions in the gel and ion electrode solution under the electric field to the minimum concentration of the protein in the sample, and then pass to the separation gel. If the accumulation gel is not present, the separation phase of the protein band becomes unclear according to the volume of the applied sample, and the separation performance is greatly deteriorated due to the molecular weight difference.

In the Lamley formulation, the ions contained in the negative electrode solution are the positive electrolytes of chlorine anions and glycine. An amphoteric electrolyte has the same number of anions and cationics in the same molecule. The degree of ionization of the amphoteric electrolyte depends on pH and becomes generally neutral at pH lower than anionic pK, electrically neutral at neutral pH, and at higher pH than cationic pK. Therefore, at low pH (6.8) in the accumulation gel, the amphoteric electrolyte loses anionicity, resulting in lower electrophoresis than the protein contained in the sample. On the other hand, the chlorine anion, which is a leading ion, moves to the anode faster than the protein with strong acidic roots and increases the electrical resistance in the gel instantaneously. Therefore, a high potential occurs in the second half to increase the moving speed of the protein and the positive electrolyte. Under basic conditions (pH 8.8) of the separation gel, the positive electrolyte recovers anionicity, so that the transfer speed is increased than that of the protein, and then the protein is moved and separated according to its inherent size into the microchannel of the gel. Therefore, for one electrophoresis, first, a separation gel having an amine partially neutralized to pH 8.8 must be hardened, and then an accumulation gel of pH 6.8 must be prepared thereon.

As such, Lamley's method requires a relatively long time (approximately 60-90 minutes) because it requires solution preparation and two copolymerization processes of different compositions, and the gel preparation requires several steps.

Therefore, in order to reduce the inconvenience of users, a specific size framework is focused on biotechnology companies such as Bio-Rad, Novex, Owl separation systems and BMA. Sells a product in which the separation gel and the accumulation gel have been polymerized in advance.

Because of the convenience of single-gel systems, several methods have been tried that do not use accumulation gels. For example, a technique for a concentration gradient gradient in which acrylamide concentration is continuously changed with respect to the electrophoretic direction, and in a gel system in which a high concentration of 20% acrylamide is continuously changed from 4 to 5%, the accumulation gel is separately It is known that it is not necessary. These techniques are disclosed in Japanese Patent Laid-Open No. 61-28512, Japanese Patent Laid-Open No. 63-210654, Japanese Patent Laid-Open No. 1-263548, and the like.

In addition, a method of preparing a separation gel using tris-phosphate buffer solution and glycerin as an electrolyte solution in a gel, and also adding agarose to a sample and dissolving it at a high temperature was applied directly to the separation gel to electrophores proteins. DeWald, DB et al, (1986) Anal. Biochem. 154, 502]. With this method, there is no need to prepare an accumulation gel.

Marshall et al. Have reported that they do not need an accumulation gel using polyacrylamide as a result of electrophoresis by using agarose directly on a separation gel and applying a sample. [Marshall, T et al, (1989) Electrophoresis 10 , 63].

However, these methods require a separate mixer device for the acrylamide concentration gradient or it is troublesome to use agarose as a kind of support in addition to acrylamide.

In Korean Patent No. 183040, as a method for increasing the resolution of the separation gel, a method of forming an electric gradient in the gel in an electric field by adding a positive electrolyte in a polyacrylamide gel has been proposed. .

The formation of the potential gradient in the gel by the addition of the positive electrolyte in the separation gel is effective to improve the resolution of the electrophoretic image according to the conventional method. However, when the gel is used as a single gel, the accumulation of the protein sample does not occur. There is an inconvenience to use.

The present invention is to solve the above problems, it is an object to provide a polyacrylamide single gel for protein electrophoresis.

Figure 1a, Figure 1b is a diagram showing the results of the electrophoresis of the whole protein and glutathione transferase (GST) fusion protein extracted from Escherichia coli (DH5α) using a polyacrylamide monogel of the present invention.

Figure 2a, Figure 2b and Figure 2c is a diagram showing the process of the accumulation of protein samples in the sample buffer solution in the protein electrophoresis method using a polyacrylamide monogel of the present invention.

Figure 3 is a protein electrophoresis method using a polyacrylamide single gel of the present invention, it is a diagram showing the results of the concentration and pH of the amine contained in the buffer solution in the separation gel on the resolution of the protein.

Figure 4 is a diagram showing the results of the protein electrophoresis method using a polyacrylamide single gel of the present invention, the concentration and composition of the positive electrolyte in the separation gel on the resolution of the protein.

The present invention relates to a polyacrylamide single gel for protein electrophoresis, by which accumulation occurs in a gel loading buffer before the protein is introduced into the polyacrylamide gel, so that a high resolution can be obtained with a single gel alone. The present invention relates to a polyacrylamide monogel for protein electrophoresis.

Specifically, the present invention is characterized in that a high resolution protein accumulation effect is achieved without adjusting gel by adjusting the concentration of amine buffer solution in polyacrylamide gel, the composition ratio of amine and amphoteric electrolyte, and pH.

That is, 1) by increasing the concentration of the amine in the separation gel to form a steep electrolyte concentration gradient with the sample buffer solution, and 2) also separating the same or different positive electrolytes contained in the negative electrolyte at the same concentration or higher This is made possible by adding to the gel the main leading ion to be an amphoteric electrolyte rather than a chloride anion.

In the present invention, the steep concentration gradient of the amine and the amphoteric electrolyte between the separation gel and the sample buffer delays the influx of protein into the separation gel. In the electric field, the inflow of the protein into the separation gel is delayed until the chlorine anion and the positive electrolyte in the catholyte accumulate the protein in the sample buffer and move to the separation gel. Subsequently, when the chlorine anion moved from the cathode electrolyte moves to the separation gel, the electrical resistance of the separation gel decreases, and the transfer of proteins begins.

In addition, when the separation gel is in a basic condition, since the separation gel does not contain chlorine anions, only the chlorine anion added to the negative electrolyte serves as the main leading ion in the separation gel, so that the potential gradient in the separation gel is added to the positive electrolyte added. It is mainly determined by. Therefore, high resolution can be realized by forming a potential gradient in the gel by the positive electrolyte.

Polyacrylamide monogel for protein electrophoresis of the present invention is characterized in that the aqueous solution containing a high concentration of amine and an amphoteric electrolyte.

In the polyacrylamide monogel for protein electrophoresis of the present invention, both the amine in the electrophoretic electrode solution and the amine in the electrolyte solution in the gel are tris (hydroxymethyl) aminomethane (hereinafter referred to as 'tris'). Is preferably.

At this time, the concentration of the amine in the gel is preferably selected from 0.3 ~ 1.0 mol / L, in any case should be higher than the amine concentration contained in the negative electrode solution and the sample buffer solution. If the concentration of the amine is 0.3 mol / L or less, no accumulation effect in the sample solution is observed, and the resolution of the protein after electrophoresis is also poor.

In the polyacrylamide monogel for protein electrophoresis of the present invention, the pH of the gel solution is in the range of 7.5-11. If the pH of the gel solution is equal to or lower than the pH of the neutral or sample solution, the accumulation effect is not good and the electrical resistance in the gel decreases, so that the moving speed of the protein is significantly different from that of the Ramley prescription gel.

In the polyacrylamide monogel for protein electrophoresis of the present invention, the amphoteric electrolyte used in the electrolyte solution in the gel is glycine, serine, asparagine, histidine, alanine, phenylalanine, glutamine, tryptophan, glutamic acid, aspartamic acid, N-tris Amino acids, such as (hydroxymethyl) methylglycine, and derivatives thereof, It is preferable that they are a kind or heterogeneous mixture chosen from these mixtures.

In the present invention, since the positive electrolyte included in the separation gel forms a gentle potential gradient in the separation gel in the electric field, it is effective in improving the resolution of protein electrophoresis similarly to the use of polyacrylamide concentration gradient gel. However, simply adding a positive electrolyte to a separation gel containing the amine concentration (0.125 mol / l) of the Lamri formulation and the amine below 0.3 mol / l, which is the lowest amine concentration of the present invention, has no effect on the efficient accumulation of protein samples and the resolution. It also appears bad.

Therefore, simply adding a positive electrolyte according to the conventional method without adjusting the concentration of the amine in the separation gel cannot realize the high resolution of the single gel.

In the present invention, the content ratio of the amine and the positive electrolyte in the separation gel is 1: 0.01 to 0.3 based on the molarity (M). When the concentration of the positive electrolyte was increased to 0.3 or more compared with the concentration of the amine, the resolution decreased markedly and the aggregation of proteins appeared. When the concentration of the positive electrolyte is lowered to 0.01 or less, it is difficult to form a potential gradient by the positive electrolyte.

In the polyacrylamide monogel for protein electrophoresis of the present invention, in order to separate proteins by molecular weight, it is preferable to use 0.01% by weight or more of dodecyl lactate (hereinafter referred to as 'SDS') in the electrophoretic electrode solution on the negative electrode side. desirable.

In the polyacrylamide monogel for protein electrophoresis of the present invention, the amphoteric electrolyte used for the electrophoretic electrode solution on the negative electrode side is particularly preferably glycine or N-tris (hydroxymethyl) methylglycine.

In the polyacrylamide monogel for protein electrophoresis of the present invention, it is preferable to use acrylamide and divinyl compound in monomer form for copolymerization of the gel support, and the divinyl compound is N, N-methylene bis acrylamide. Is preferably.

In the polyacrylamide monogel for protein electrophoresis of the present invention, as a polymerization initiation means, peroxides such as ammonium persulfate (hereinafter referred to as 'APS') and N, N, N ', N',-tetramethylethylenediamine It is preferable to use a redox-type polymerization initiator using a reducing agent such as (hereinafter referred to as 'TEMED') as a polymerization catalyst. However, it is not specifically limited to these, The photoinduction copolymerization reaction with riboflavin can be used.

In the present invention, since the SDS is not added to the gel, both the protein electrophoresis method in the natural state and the electrophoresis method under denaturing conditions may be possible depending on the presence or absence of SDS in the sample treatment solution and the electrophoretic negative electrode solution.

Hereinafter, a method for preparing a polyacrylamide gel for protein electrophoresis according to the present invention will be described in detail through examples. However, the present invention is not limited to these examples.

Example 1: raemri polyacrylamide gel and compared with a single protein electrophoresis gel of the present invention

The electrophoresis kit sold by Hopper was purchased, and an electrophoretic device using a rectangular glass plate having a width of 10 cm and a length of 8 cm and a spacer having a thickness of 0.75 mm was assembled.

10 wt% acrylamide concentration, 0.1 wt% APS and 0.04 wt% TEMED were added to the electrolyte solution, and then injected between the two glass plates and polymerized for 30 minutes to obtain a gel for electrophoresis. At this time, the electrolyte solution used was 0.7 mol / L of tris (pH 10.5), 0.1 mol / L glycine, and 0.1 mol / L serine.

E. coli (DH5α) total protein extract was applied to the gel by volume of 1 ~ 15μl, bovine serum albumin (BSA) and glutathione transferase (GST) fusion protein was applied. The sample buffer solution ((beta) -mercaptoethanol, SDS, glycerin, bromine phenol blue) of the Ramley prescription was previously added to the protein sample, and it boiled at 95 degreeC for 4 minutes, and it electrophores. The gel was 0.75 mm thick and 10 uneven wells were used, and 2-15 μl of sample was applied to each well.

The composition of the electrophoretic electrode solution was Lamley formulation at a tris concentration of 0.025 mol / l, glycine concentration of 0.192 mol / l, and SDS 0.1 wt%.

In order to compare the separation and mobility of the proteins in the sample with the conventional discontinuous gel system, gels were prepared according to the Ramley prescription, and electrophoresis was performed under the same conditions by applying the same volume of the sample. In addition, only the separation gel was prepared in the Lamley formulation to prepare the same sample, and electrophoresis was performed under the same conditions.

Electrophoresis was performed at a constant voltage of 140V, and the electrophoresis was stopped when the electrophoretic end of the bromine phenol blue pigment was 5 mm from the bottom.

After electrophoresis, the proteins were stained with a dye solution containing Coomassie brilliant blue R250 for 1 hour and decolorized with a destaining solution containing methanol / acetic acid.

Experimental results are shown in Figures 1a and 1b, Figure 1a is a photo of the dye stained with a dye solution after electrophoresis of the protein, Figure 1b is a result of the analysis of the separation phase of the protein by analyzing the rectangular region in the gel picture to be.

1A and 1B, in the case of a 0.75 mm gel and 10 uneven wells, a maximum of about 15 μl of sample can be electrophoresed. In the gel system of the present invention, the application and electrophoresis of the sample up to about 15 μl are similarly performed. It was possible. In addition, there was no change in resolution and movement distance for each protein with respect to the concentration and volume of the applied protein extract.

When the protein was electrophoresed using only the separation gel in the Lamley gel system, it was confirmed that the protein was not efficiently separated.

When electrophoresis was performed with the polyacrylamide monogel of the present invention, there was no aggregation of protein observed in the electrophoresis by Lamley's formulation and a separation phase similar to that when using a continuous concentration gradient gel was obtained.

Example 2 Effect of Concentration of Amine on Accumulation of Protein Samples

To determine the effect of the concentration of the amine contained in the buffer solution in the separation gel on the accumulation of protein samples, electrophoresis was performed while varying the amount of protein samples. At this time, all the experimental conditions were used in the same manner as in Example 1.

As a control of the experiment was used a single gel containing 0.075 mol / L Tris, 0.1 mol / l glycine, 0.1 mol / l serine, the final pH was adjusted to 7.5 with hydrochloric acid.

Each well was subjected to the same amount of E. coli protein extract in a volume of 1 ~ 15μl, followed by electrophoresis.

The results of electrophoresis are shown in Figs. 2a, 2b and 2c.

According to Figure 2a, in the control group containing a low concentration of amine was not observed accumulation in the buffer containing the protein sample, when using the polyacrylamide single gel of the present invention that the accumulation of protein effectively progressed I could confirm it. The arrow indicates the boundary between the catholyte buffer and the polyacrylamide gel.

According to Figure 2b, the control of the addition of the positive electrolyte to the low concentration of the amine did not improve the resolution of the protein, it can be seen that more dependent on the amine concentration when the amount of protein is small.

Figure 2c is a diagram showing the results of the electrophoresis of the same amount of E. coli extract with varying volumes to 1 ~ 15μl, while the resolution of the protein is not good in the control group added with a low concentration of amine, polyacrylamide according to the present invention In the single gel it was confirmed that the separation phase of the protein is clear. In addition, the amount of protein was the same, the volume of the sample was confirmed that the protein accumulation is not good.

Example 3: Influence of pH and concentration of the amine on the resolution of the proteins

To determine the effect of the concentration and pH of the amine contained in the buffer solution in the separation gel was electrophoresed while varying the concentration or pH of the amine in the separation gel. At this time, all the experimental conditions were used in the same manner as in Example 1.

The electrophoresis results are shown in FIG. 3, and the higher the amine concentration in the separation gel was, the clearer the separation phase was. The higher the pH of the separation gel was, the better the separation state of the protein was.

Example 4: Effects of the concentration and composition of the electrolyte on the positive resolution of proteins

In order to determine the effect of the composition of the positive electrolyte in the separation gel on the protein's separation ability, the electrophoresis was performed by varying the concentration and composition of the positive electrolyte added to the separation gel. At this time, all the experimental conditions were used in the same manner as in Example 1 except for changing the concentration of the positive electrolyte.

As shown in FIG. 4, a more clear separation phase was obtained when a mixture of different types of positive electrolytes was used than when a single positive electrolyte was added to the separation gel. However, no significant improvement in separation was observed even by increasing the concentration of the positive electrolyte. In addition, it was confirmed that aggregates of proteins are formed when the concentration of the positive electrolyte is high.

According to the polyacrylamide monogel for protein electrophoresis of the present invention, it is possible to exhibit high resolution without using an accumulation gel and obtain an effect similar to that of a conventional discontinuous gel.

In addition, the polyacrylamide single gel for electrophoresis of the present invention can be used in a low-cost production process in the production of the polyacrylamide polymerized gel because the production method is simple and easy to use.

Claims (12)

The polyacrylamide gel is an aqueous solution containing an amine and an amphoteric electrolyte, the concentration of the amine is 0.3-1.0 mol / l, and the molar concentration ratio of the content of the amine and the amphoteric electrolyte is 1: 0.01-0.3. Polyacrylamide Monogel The polyacrylamide monogel for protein electrophoresis according to claim 1, wherein the pH of the electrolyte solution in the polyacrylamide gel is in the range of 7.5 to 11. The polyacrylamide monogel for protein electrophoresis according to claim 1 or 2, wherein the amine in the polyacrylamide gel is tris (hydroxymethyl) aminomethane. The amphoteric electrolyte used for the electrolyte solution in the polyacrylamide gel according to claims 1 to 3 is glycine, serine, asparagine, histidine, alanine, phenylalanine, glutamine, tryptophan, glutamic acid, aspartamic acid, N-tris (hydride). Amino acids such as oxymethyl) methylglycine and derivatives thereof, polyacrylamide monogel for protein electrophoresis, characterized in that it is a kind selected from mixtures thereof [5] The polyacrylamide monogel for protein electrophoresis according to claim 4, wherein the heterogeneous mixture is selected in the amphoteric electrolyte. The polyacrylamide monogel for protein electrophoresis according to any one of claims 1 to 5, comprising acrylamide and divinyl compounds in monomer form for copolymerization of the polyacrylamide gel support. The polyacrylamide monogel for protein electrophoresis of claim 6, wherein the divinyl compound is N, N-methylene bis acrylamide. 8. The polyacrylamide monogel for protein electrophoresis according to claim 1, wherein the polymerization initiation means comprises a redox-type polymerization initiator. The polyacrylamide monogel for protein electrophoresis according to claim 8, wherein the polymerization starter comprises ammonium peroxide and N, N, N ', N', tetramethylethylenediamine. Protein electrophoresis method for separating proteins using the polyacrylamide monogel for protein electrophoresis of claim 1 The protein electrophoresis method according to claim 10, wherein at least 0.01% by weight of dodecyl lactate is used as the electrophoretic electrode solution on the cathode side. 12. The protein electrophoresis method according to claim 10, wherein the amphoteric electrolyte used for the electrophoretic electrode solution on the cathode side is glycine or N-tris (hydroxymethyl) methylglycine.