KR101111351B1 - Pre-stained protein size marker based on synthetic nonionic hydrophilic polymers - Google Patents

Abstract

The present invention relates to prestained protein size markers, and more particularly to prestained protein size markers based on synthetic nonionic hydrophilic polymers. According to the method proposed in the present invention, synthetic nonionic hydrophilic polymers, which are more easily secured and have a simple molecular structure, can be used as raw materials instead of proteins commonly used as raw materials for pre-stained size markers. It is easy to secure the raw material of the pre-stained protein size marker and control the manufacturing process.

Prestained Protein Size Markers, Synthetic Nonionic Hydrophilic Polymers

Description

Pre-stained protein size marker based on synthetic nonionic hydrophilic polymers

The present invention relates to prestained protein size markers, and more particularly to prestained protein size markers based on synthetic nonionic hydrophilic polymers.

Protein size markers are aimed at estimating the size of a target protein on gels in electrophoresis, which is typically performed for identification and analysis of proteins in all bio-related research and development. It is used as a product. Initially developed protein size markers were used to visualize the target protein and protein size markers together by staining the protein in the gel after electrophoresis. Prestained protein size for later use as a tool for monitoring the real-time progress of electrophoresis during electrophoresis in addition to providing convenience in estimating the size of the target protein Marker (pre-stained protein size marker) has been developed. Unlike unstained protein size markers (unstained protein size markers), prestained protein size markers are stained with chemicals (ie dyes) that color the proteins that make up the protein size marker. Therefore, the prestained protein size markers have their own color even without a separate staining process, so that individual constituent bands are visible from the electrophoresis. These prestained protein size markers, in addition to the real-time observation of electrophoresis during electrophoresis, can be transferred from gel to membrane in an electro-transfer step in western blotting using electrophoretic gels. It is also used to estimate the degree of protein transfer.

Conventional pre-stained protein size markers are prepared using the protein as raw material, as in conventional protein size markers. These raw proteins are extracted and used in nature, or more effectively, they are produced through recombinant protein expression and then separated and purified. In general, since the protein size marker is composed of protein bands of various sizes, in order to provide such protein bands of various sizes, proteins of various sizes must be used as raw materials.

In the case of proteins, different sizes are possible only if the types are different. For this reason, several protein raw materials used to prepare conventional protein size markers had to be used for the production of one protein size marker. To prepare these different kinds of proteins, each protein had to be subjected to a production and separation / purification process suitable for that protein, and the burden was considerable. In particular, since the performance of the final protein size marker is highly dependent on the level of purification of the raw protein, the degree of purification of these raw proteins is very important. By the way, purifying proteins from cell cultures or natural sources is a very difficult task since they contain a variety of other proteins from the beginning. This typically requires a lot of money and time. In addition, the recovery / recovery of specific proteins is not high, since it is common for complex and difficult processes to separate / recover specific proteins from them.

On the other hand, since proteins are basically polymers of various kinds of amino acids, there are various kinds of functional groups to which dyes can be added depending on proteins, and the number of them varies widely. Even if the same dye is added to the protein due to the difference in the functional group of each protein, the result is inevitable and should be applied after setting appropriate conditions for each protein. It is also undesirable from the standpoint of dye addition reaction control that the number of functional groups varies between proteins and that the number is quite large per molecule of protein. Due to the difference in the reaction conditions, the number of dyes bound to one molecule of protein varies greatly, and as a result, the protein / dye complex to which the dye is added varies even if the same protein is started. In the conventional case of using a protein as a raw material of a prestained protein size marker, dyes are randomly bound to the whole protein. In this case, there is a limit in obtaining only a uniform type (that is, a constant number of dyes bound per molecule of protein), and usually several kinds of protein / dye complexes coexist. These different complexes eventually lead to more spreading of the bands of protein size markers because of their differing behavior in electrophoresis.

In addition, in order to efficiently add dyes to proteins, the inherent conformation of proteins must be released. This is called protein denaturation. Such protein denaturation can be implemented in a variety of ways, but with regard to protein size markers, denaturation methods using surfactants and heat for protein denaturation are widely used. However, because proteins are quite complex substances and their kinds vary greatly, the degree of degeneration of each protein is different. The degree of denaturation of these proteins must be consistently achieved so that the result of the dye addition reaction using them can be constant. However, controlling the degree of protein denaturation in reality is very difficult in reality.

In order to solve the drawbacks of using these proteins as raw materials for the preparation of pre-stained protein size markers, it is necessary to develop pre-stained protein size markers based on new materials completely different from proteins.

The present inventors have tried to find a protein-free non-protein material as a raw material for the production of pre-stained protein size markers. The invention has been completed.

Throughout this specification, numerous papers and patent documents are referenced and their citations are indicated in parentheses. The disclosures of cited papers and patent documents are incorporated by reference into the specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

Therefore, an object of the present invention is to solve the problems of the prior art and the technical problem that has been requested from the past.

It is an object of the present invention to provide a method for producing prestained protein size markers based on synthetic nonionic hydrophilic polymers.

It is still another object of the present invention to provide a prestained protein size marker based on synthetic nonionic hydrophilic polymers.

In order to solve the problems of the present invention as described above, the present invention provides a prestained protein size marker based on a synthetic nonionic hydrophilic polymer among non-protein components and a method for producing the same.

The synthetic nonionic hydrophilic polymer may be selected from various synthetic polymers according to a method that can be easily carried out by those skilled in the art, but is not limited thereto. , Polypropylene glycol, polyethylene propylene glycol, polybutylene glycol, polypropylene butylene glycol, and polyethylene oxide is preferably selected from the group consisting of. In particular, polyethylene glycol is the most preferred since it is already widely used for various purposes in the art and has a wide range of familiar, commercially available molecular weights, and an oxyethylene skeleton has hydrophilic properties suitable for application of the present invention. can do.

The synthetic nonionic hydrophilic polymers have a common feature of hydrophilicity, and although they are neutral polymers without charge, they are classified under normal electrophoresis conditions (ie, standard Laemmli SDS-PAGE conditions). It can react with ionic surfactants such as sodium dodecyl sulfate to form a complex with negative charge (J. Phys. Chem. B 108: 8960-8969 , 2004). These negatively charged complexes can migrate in gels in the electric field of electrophoresis (Electrophoresis 28: 2801-2807, 2007). The present invention utilizes these properties. In addition to SDS, other ionic surfactants may exhibit the above effect, but in general electrophoresis, SDS is widely used as a surfactant, and thus, SDS is preferable unless there is a specific reason.

The synthetic nonionic hydrophilic polymers may be linear or branched in structure. Since the branched form is also a kind of complicated structure, the linear is the most preferable structure of the polymer in consideration of the ease of manufacture and the like.

In order to use the synthetic nonionic hydrophilic polymer as a raw material of the protein size marker, the synthetic nonionic hydrophilic polymer should be prepared to have several different sizes (molecular weights) suitable for the various bands constituting the protein size marker. For synthetic nonionic hydrophilic polymers, there is no need to change their type to achieve this, as with proteins. By controlling only its degree of polymerization, polymers of different molecular weights composed of the same monomer can be prepared. This can be easily carried out by those skilled in the art of polymer synthesis, so a detailed description thereof will be omitted.

However, synthetic nonionic hydrophilic polymers to be used as raw materials of the respective bands should have a molecular weight distribution in the narrow range as much as possible. In general, it is difficult to control the degree of polymerization completely in the production of polymers, so there is a slight molecular weight difference between the polymers synthesized in one reaction. This is called polydispersity. In order to implement the present invention smoothly, there is no particular limitation on the degree of tolerance, but the narrower the distribution range of the molecular weight, the better. The narrower the molecular weight distribution (the lower the polydispersity), the less the spread of the corresponding bands in the pre-stained protein size markers that are finally produced using this as a raw material, resulting in better performance of the pre-stained protein size markers. The performance of prestained protein size markers is the sharpness and sharpness of the bands. As the band spreads, the performances fell. In recent years, the polymer synthesis technology has been greatly advanced, and the range of the molecular weight size of the synthetic polymer produced can be controlled very narrowly. Since the molecular weight control technology of the polymer to be synthesized can be easily carried out by those skilled in the art of polymer synthesis, a detailed description thereof will be omitted.

In order to prepare prestained protein size markers, dyes must be bound to the synthetic nonionic hydrophilic polymers. For this purpose, since the general synthetic nonionic hydrophilic polymer itself is inferior in reactivity with the dye, a functional group capable of reacting with the dye should be introduced into the synthetic nonionic hydrophilic polymer. This process is called activation. The activation can be selected at the target position anywhere in the middle or at the end of the synthetic nonionic hydrophilic polymer. However, for practical implementation the terminal is the best introduction position for manufacturing reasons. Activation implemented by introducing such chemical functional groups can be easily performed by those skilled in the art of organic chemistry, and thus a detailed description thereof will be omitted.

For reference, a brief description of a method of activating polyethylene glycol (PEG) can be used to synthesize derivatives having a single functional group from methoxy polyethylene glycol (Pharmaceutical Science & Technology Today 1: 352-356, 1998). . By replacing one hydroxy group of polyethylene glycol with a methyl ether group and bonding a functional group containing an electrophile to the other hydroxy group, polyethylene glycol having only one terminal activated (Pharmaceutical Science & Technology Today 1: 352-356, 1998). Activation at both ends is similar.

Even when a functional group is introduced at the terminal of the synthetic nonionic hydrophilic polymer, it may be introduced at both ends or only at one end. When using a synthetic nonionic hydrophilic polymer having a functional group only at one end as a raw material, the dye addition reaction is easier to control because there is only one position where dye can be added (ie, whether dye is added or not). There is only one dye that can be added per molecule of synthetic nonionic hydrophilic polymer. As a result, when using the same dye, the degree of color development is weaker than when two dyes are added. have. When using a synthetic nonionic hydrophilic polymer having an activation group at both ends as a raw material, since there are two positions where dye can be added, it is difficult to control a dye addition reaction, thereby obtaining a constant product. For the purposes of this study, there are disadvantages in that there are more variables (ie, when one dye is added, two dyes are added, and three dyes are not added). Since the number of dyes that can be added per molecule is large compared to the case where only one end is used, more powerful color development is possible. Because of these mutual strengths and weaknesses, the application of chemical functional groups for the activation of synthetic nonionic hydrophilic polymers depends entirely on the choice of practitioner, whether it is applied to one end or both ends.

For reference, in order to further enhance the color development, a dye donor prepared by combining a plurality of dyes in advance with a polymer may be added at one end at an end (en bloc). In this case, even though one end of the synthetic nonionic hydrophilic polymer is used, much enhanced color development can be obtained than when only one dye is added. Such techniques are well known in the dye-related art and therefore detailed descriptions thereof are omitted.

Synthetic nonionic hydrophilic polymers into which functional groups are introduced through such activation are commonly referred to as derivatives of synthetic nonionic hydrophilic polymers. Such derivatives can be prepared in a wide variety of ways using known methods. Instead of omitting detailed descriptions, some representative examples of activated polyethylene glycol derivatives will be presented. Polyethylene glycol derivatives include PEG-N-hydroxysuccinimide-activated esters, PEG-epoxide, PEG-tresylate, PEG-carbonyl imidazole, PEG-nitrophenyl carbonates, PEG-aldehydes, PEG-amines, PEG-isocyanates -isocyanate), PEG-hydrazide and the like.

As a dye that can be used for dye addition to the activated synthetic nonionic hydrophilic polymer, both general dyes and fluorescent dyes can be used. Examples of common dyes that can be used include, but are not limited to, Caledon SF, Cassulfon, Indigo, Imperon, Levafix, Procion, Remazol, Sirius (trade name), and the like. Fluorescent dyes also include but are not limited to Dansyl, Fluorescein, Texas Red, Cascade Blue, Cascade Yellow, Erythrosin, Coumarin, NBD, Pacific Blue, PyMPO, Pyrene, Phycoerythrin, Cy2, Cy3, Cy5, Cy7, NBD-Phallacidin ( The above is a brand name).

In order to add such a dye to the derivative of the activated synthetic nonionic hydrophilic polymer, the dye must also be prepared in a form having a functional group capable of binding to an active site of the synthetic nonionic hydrophilic polymer. To this end, dyes are prepared in the form of various derivatives. For example, derivatives in the form of succinimidyl esters, isothiocyanates, carboxylic acids, sulfonyl chlorides and the like may optionally be used. The preparation of such derivatives can also be easily carried out by those skilled in the art of organic chemistry, and thus a detailed description thereof will be omitted.

However, derivatives of these dyes should not be prepared in any form, but must be in a form capable of reacting with a synthetic nonionic hydrophilic polymer derivative. This can be naturally determined when the type of chemical bond to be used for bonding the dye with the synthetic nonionic hydrophilic polymer derivative is determined. This can be easily carried out by those skilled in the art of organic chemistry, so a detailed description thereof will be omitted. For reference, various dye derivatives are sold commercially. Therefore, you may select the appropriate one.

Chemical reactions that can be used to bond synthetic nonionic hydrophilic polymer derivatives with dye derivatives include, for example, condensation, acylation, esterification, and reductive alkylation. It can be achieved by many organic chemical reactions such as alkylation). This can be easily carried out by those skilled in the art of organic chemistry, so a detailed description thereof will be omitted.

The present invention provides a technique that can replace the production of a pre-stained protein size marker based on a conventional protein material, and secures the raw material suitable for mass production of a pre-stained protein size marker in an easier way than the conventional technology It can provide a means to do it. In addition, the material constituting each band of the prestained protein size marker can be prepared more uniformly, thereby improving the performance of the final prestained protein size marker. Specifically, by using a synthetic nonionic hydrophilic polymer according to the present invention in place of a conventional protein, a protein denaturation step can be eliminated, and access to a position to which a dye will be added becomes easier as compared to the case where a protein is used. The efficiency is improved, because there are multiple dye addition sites and the chemical functionalities that make up these additional positions provide a certain limited number of dye addition sites compared to the use of various proteins and in addition the reactivity of the constituent chemical functionalities is the same The dye addition reaction can be easily controlled, and the separation / purification of the dye-added complex is facilitated. In particular, industrial use can be expected in consideration of the fact that prestained protein size marker is the most commonly used product in bio-related research and development, and the research atmosphere in which the use of prestained protein size marker is becoming common.

As described above, the present invention provides a prestained protein size marker based on a synthetic nonionic hydrophilic polymer which is a non-protein material.

Hereinafter, the content of the present invention will be described in more detail, but it will be understood by those skilled in the art that the present invention may be variously modified and changed without departing from the spirit and scope of the present invention.

In the following examples of the present invention, PEG-amine is used as an activated form of a synthetic nonionic hydrophilic polymer, a Remazole dye is used as a dye, and an amino group and vinyl sulfone are used to bond the synthetic nonionic hydrophilic polymer derivative and the dye. Although condensation reactions between (vinyl-sulfone) groups are utilized, this is only one example provided for understanding the present invention, and the present invention is not limited thereto.

For reference, in the following examples, a solution in which a dye is added to a synthetic nonionic hydrophilic polymer derivative and then separated / purified is referred to as a “primary polymer / dye complex solution” for convenience. A solution prepared by mixing the "primary polymer / dye complex solution" is referred to as "secondary polymer / dye complex solution", and the sample loading using the "secondary polymer / dye complex solution" thus prepared in conventional electrophoresis Solutions prepared by mixing with sample loading buffer will be referred to as “prestained protein size marker solution”.

Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples are merely illustrative of the present invention, and the scope of the present invention is not limited to these Examples.

Example  1: synthetic Nonionic  Combination of Hydrophilic Polymer Derivatives and Dye Derivatives

Commercially available PEG-amine (molecular weight: 20,000; P1AM-20, Sunbio, Korea) was purchased and used as a synthetic nonionic hydrophilic polymer derivative. The PEG-amine was dissolved in 125 mM Na 2 CO 3 buffer at pH 11 to a final PEG concentration of 20 mg / ml. This will be referred to as "PEG solution". Dye derivatives include vinyl resulfone reactive dye, Remazol Brilliant Blue R special (purchased from DyStar), Remazol Brilliant Orange 3R spec gran (purchased from Dystar), and Remazol Brilliant Red F3B gran (purchased from Dystar). And Uniblue A sodium salt (purchased from Sigma) were used, each of which was finally dissolved in water to 400 mg / ml. The solution thus prepared will be referred to as a "dye solution". The PEG solution prepared above and each dye solution were mixed at a volume ratio of 5: 1, and left to stand in a 60 ° C thermostat for 30 minutes to allow dye derivatives to be added to PEG-amine. After the addition reaction was completed, it was taken out of the thermostat and left at room temperature for 20 minutes to cool the reaction solution to room temperature. The solution of the binder to which the dye was added to the PEG derivative thus obtained was prepared.

Example  2: Preparation of Primary Polymer / Dye Complex Solution

The solution of the binder to which the dye was added to the PEG derivative prepared in Example 1 contained many unreacted dye derivatives which remained unreacted. A "primary polymer / dye complex solution" can be obtained by removing unreacted dye derivative from a solution of a binder in which dye is added to the PEG derivative. Various separation and purification methods can be used for this, but gel-filtration, which is a method widely used in the art, is most easily used. Such gel-filtration is a known technique that is utilized for various purposes in chemistry and biology. The gel-filtration method used in this example is briefly shown as follows. Commercially available Sephadex G-25 (Amersham Bioscience) was purchased and suspended in water to a final 10% (w / v). This was enough to swell Sephadex G-25 powder for one day at room temperature or refrigerated. It was then used while refrigerated. In the actual separation step, the Sephadex G-25 suspension was poured into a spin-column tube (Prober, Intron Biotechnology) and used after removing water through centrifugation. Thus, 600 μl of the solution of the conjugate to which the dye was added to the PEG derivative prepared in Example 1 was added to the Sephadex G-25 column from which water was removed, and this was centrifuged at 400 × g for 2 minutes. The filtrate thus obtained was added to a new Sephadex G-25 column and further centrifuged. This step was repeated even after centrifugation until the packed matrix of the Sephadex G-25 column had almost no color of the dye. Through this, a "primary polymer / dye complex solution" was obtained.

Example  3: prestained protein size Marker  Preparation of the solution

"Pre-stained protein size marker solution" was prepared using the "primary polymer / dye complex solution" obtained in Example 2. To this end, each of the “primary polymer / dye complex solutions” obtained in Example 2 had the same volume of sample loading buffer (4% SDS, 125 mM Tris-HCl, 10 mM EDTA, 100 mM DTT, 60% glycerol, pH 6.8). Several kinds of "pre-stained protein size marker solution" were prepared by adding the same. In this case, the sample loading buffer used is twice the concentration of the sample loading buffer used in the art, and when the same amount of the sample loading buffer is mixed with the “primary polymer / dye complex solution”, the sample loading usually used in the art is finally obtained. It will be equal to the concentration of the buffer. For reference, it is very natural in the art to change some of the compositions in the sample loading buffer.

Example  4: Investigate performance by electrophoresis

The performance of the pre-stained protein size marker solution prepared in Example 3 was examined through general electrophoresis, an experimental method used for protein analysis. Electrophoresis was performed using a 12% SDS-PAGE gel, and electrophoresis buffer used in electrophoresis was used as Tris-Glycine buffer, which is usually used in electrophoresis for protein analysis. The result of using Remazol Brilliant Blue R special as a dye derivative is shown in FIG. 1, and the result of using Remazol Brilliant Red F3B gran as a dye derivative is shown in FIG. 2, and the result of using Remazol Brilliant Orange 3R spec gran as a dye derivative. 3, the results of using Uniblue A sodium salt as a dye derivative are shown in FIG.

From the results of Figures 1 to 4, it can be seen that the binding between the desired PEG and the dye was achieved first, and the prestained protein size markers prepared according to the method of the present invention are usually protein based prestained protein size. As with the marker, it can be seen that the electrophoresis can be moved. In order to be used as a prestained protein size marker, the most important point is that the electrophoresis for protein analysis purposes must be able to move in the same direction as the protein to be analyzed.

Example  5: Investigate performance in electrophoresis

As a dye derivative prepared in Example 3, an electrophoresis method performed after electrophoresis, which is an experimental method in which “prestained protein size marker solution” prepared using Remazol Brilliant Blue R special, is commonly used for protein analysis, is performed. -transfer) to examine the performance. Electrophoresis is a key step in a general method called membrane blotting, which refers to the transfer of proteins separated from the gel to the membrane under an electric field by electrophoresis. In the electrophoresis method used in the present embodiment, a widely used polyvinylidene fluoride (PVDF) membrane was used, and a conventional transfer buffer (2.9 g / L glycine, 5.8 g / L Tris, 0.37 g / L SDS, 200) was used. ml / L methanol) was used for 2 hours at 300 mA. The results are shown in FIG.

From the results of FIG. 5, it was confirmed that the prestained protein size markers prepared according to the method of the present invention were moved from the electrophoretic gel to the membrane by electrical force as desired. It was also confirmed that the polymer / dye complex of the moved prestained protein size marker binds well to the membrane. From this, it can be seen that the prestained protein size marker prepared according to the method of the present invention can be effectively used for membrane blotting.

Example  6: Prestained protein size with several polymer / dye complexes Marker

According to the method of Example 1 to Example 3, prestained protein size markers including polymer / dye complexes having various sizes were prepared while varying the molecular weight of the PEG used. According to the method described in Examples 1 and 2, two PEG-amines having a molecular weight of 12 kDa and 20 kDa were respectively stained with Remazol Brilliant Blue R special or Remazol Brilliant Orange 3R spec granules, respectively. Primary polymer / dye complex solution ”. Among these four types of "primary polymer / dye complex solution", "primary polymer / dye complex solution" prepared using Remazol Brilliant Blue R special as a dye derivative was prepared from 3.5: 2.4 (20kDa PEG-amine by volume ratio). “Primary polymer / dye complex solution”: “Primary polymer / dye complex solution” prepared from 12 kDa PEG-amine) to mix “secondary polymer / dye complex solution” containing two PEG / dye complexes Prepared. Meanwhile, the "primary polymer / dye complex solution" prepared by using Remazol Brilliant Orange 3R spec gran as a dye derivative in these four types of "primary polymer / dye complex solution" was used in a volume ratio of 3.5: 2.4 (20 kDa PEG-amine). "Primary polymer / dye complex solution" prepared from: "primary polymer / dye complex solution" prepared from 12 kDa PEG-amine) and another "secondary polymer containing two PEG / dye complexes" / Dye complex solution ". This was mixed with eastern blood sample loading buffer as in Example 3 to prepare two kinds of "pre-stained protein size marker solution". The result of investigating this in the same manner as in Example 4 is shown in FIG. 6.

In addition, a "pre-dyed protein size marker solution" was prepared by mixing the "primary polymer / dye complex solution" prepared using the above-described different dye derivatives. The "primary polymer / dye complex solution" prepared by dyeing 12 kDa PEG using Remazol Brilliant Blue R special as a dye derivative and the "primary" prepared by staining 20 kDa PEG using Remazol Brilliant Orange 3R spec granule as dye derivative The polymer / dye complex solution ”was mixed at a volume ratio of 3.5: 2.4 to prepare a“ secondary polymer / dye complex solution ”. This was mixed with eastern blood sample loading buffer to prepare a “pre-stained protein size marker solution”. Electrophoresis results of this investigation in the same manner as in Example 4 are shown in FIG. 7.

From this it can be seen that according to the method of the present invention it is possible to prepare a "pre-stained protein size marker" having a band of various sizes.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is a result of the electrostaining of the prestained protein size marker prepared according to the present invention using Remazol Brilliant Blue R special as a dye derivative. Lane M is a protein based prestained protein size marker and lanes 1-4 are the result of loading similar amounts of prestained protein size markers prepared according to the present invention. The actual loading amount was used in excess for accurate identification.

FIG. 2 shows the results of electrophoresis of prestained protein size markers prepared according to the method of the present invention using Remazol Brilliant Red F3B gran as a dye derivative. Lane M is a protein based prestained protein size marker, and lanes 1 and 2 are the result of loading different amounts of gel onto the prestained protein size markers prepared according to the invention. The actual loading amount was used in excess for accurate identification.

Figure 3 shows the results of electrophoresis of prestained protein size markers prepared according to the method of the present invention using a Remazol Brilliant Orange 3R spec gran as a dye derivative. Lane M is a protein based prestained protein size marker and lanes 1-4 are the result of loading different amounts of gel onto the prestained protein size markers prepared according to the invention. The actual loading amount was used in excess for accurate identification.

FIG. 4 shows the results of electrophoresis of prestained protein size markers prepared according to the method of the present invention using Uniblue A sodium salt as a dye derivative. Lane M is a protein based prestained protein size marker and lanes 1-4 are the result of loading different amounts of gel onto the prestained protein size markers prepared according to the invention. The actual loading amount was used in excess for accurate identification.

FIG. 5 shows the result of dye pre-stained protein size markers prepared according to the method of the present invention using Remazol Brilliant Blue R special as a dye derivative, followed by electrophoresis and transfer to the membrane. The prestained protein size marker of the invention was transferred to the membrane by electrophoresis using a gel prepared by electrophoresis of the prestained protein size marker of the invention.

Figure 6 is the result of examining the prestained protein size marker (containing two PEG / dye complexes) of the present invention by electrophoresis. (A) Prestained protein size markers prepared by staining each with Remazol Brilliant Blue R special using two PEG-amines, 12kDa and 20kDa. (B) Prestained protein size markers prepared by staining each with Remazol Brilliant Orange 3R spec gran using two PEG-amines of 12 kDa and 20 kDa. In both Figures 6 (A) and (B), lane M is a protein based prestained protein size marker, and lane 1 is the result of loading the gel with a prestained protein size marker prepared according to the present invention.

7 is a result of examining the prestained protein size markers (complexes of different dyes in two kinds of PEG) of the present invention by electrophoresis. PEG-amine 12kDa was stained with Remazol Brilliant Blue R special and PEG-amine 20kDa was stained with Remazol Brilliant Orange 3R spec granules. Lane M is a protein based pre-stained protein size marker, and lane 1 is the result of loading the gel with a pre-salt colored protein size marker prepared according to the method of the present invention.

Claims (9)

Prepared by chemically bonding a dye to a nonionic, hydrophilic, non-protein synthetic polymer selected from the group consisting of polyethylene glycol, polypropylene glycol, polyethylenepropylene glycol, polybutylene glycol, polypropylene butylene glycol, and polyethylene oxide One pre-stained protein size marker. delete delete The prestained protein size marker according to claim 1, wherein the dye is selected from the group consisting of a general dye, a fluorescent dye, and a dye donor prepared by combining a plurality of dyes with a polymer in advance. Addition reaction of nonionic and hydrophilic non-protein synthetic polymers and dye derivatives selected from the group consisting of polyethylene glycol, polypropylene glycol, polyethylenepropylene glycol, polybutylene glycol, polypropylene butylene glycol, and polyethylene oxide step; Removing unreacted dye derivative from the polymer / dye complex solution produced by the addition reaction; A method of preparing a prestained protein size marker based on a nonionic, hydrophilic, non-protein-based synthetic polymer, comprising: mixing a solution from which an unreacted dye derivative is removed with a sample loading buffer. The method of claim 5, wherein mixing the unreacted dye derivative-free solution with the sample loading buffer further comprises mixing one or more solutions from which the unreacted dye derivative has been removed. A method for preparing prestained protein size markers based on hydrophilic non-protein based polymers. delete delete The nonionic, hydrophilic, non-protein-based system of claim 5, wherein the dye is selected from the group consisting of a general dye, a fluorescent dye, and a dye donor prepared by combining a plurality of dyes with a polymer in advance. Method for preparing prestained protein size markers based on synthetic polymers.

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