WO2008065986A1

WO2008065986A1 - Method of isoelectric point separation and method of determining hydrogen ion concentration gradient in the separation field

Info

Publication number: WO2008065986A1
Application number: PCT/JP2007/072739
Authority: WO
Inventors: Toru Sano
Original assignee: Nec Corporation
Priority date: 2006-12-01
Filing date: 2007-11-26
Publication date: 2008-06-05
Also published as: JP2008139168A

Abstract

A method of determining a pH gradient in which the pH distribution in separation field can be accurately determined on real-time basis without disturbing judgment of sample separation results and without needing complex operation; and a method of isoelectric point separation using the above method. There is provided a method of determining a pH gradient in separation field for isoelectric point separation in which the pH gradient is determined in a separation field for isoelectric point separation containing a carrier ampholyte, which method comprises the step of, using a carrier ampholyte containing multiple fluorescent components with known isoelectric points different from each other, determining the pH gradient with the use of the fluorescent components as isoelectric point markers, or measuring the fluorescence intensity distribution along the separation field; and the step of determining the pH gradient in separation field from known isoelectric points and peak making positions of the fluorescence intensity distribution. Further, there is provided a method of isoelectric point separation, comprising the step of finding the isoelectric point of analyte with the use of the pH gradient determined by the above method.

Description

Specification

Isoelectric point separation method and hydrogen ion concentration gradient determination method in the separation field

Technical field

[0001] The present invention relates to a method for determining a hydrogen ion concentration (pH) gradient in a separation field at the time of isoelectric point separation used for protein analysis or the like.

Background art

[0002] When a mixture of amphoteric components such as proteins and peptides is isoelectrically separated, each component converges and is separated at an isoelectric point specific to each component. At this time, the hydrogen ion concentration (pH) at which the charge is apparently zero is an isoelectric point, and a solid such as polyacrylamide or agar or a liquid such as one in a pillar is used as the separation field. By knowing how the pH distribution is occurring in the separation field, the isoelectric point can be determined in the case of unknown isoelectric point components, and in the case of known isoelectric point components. You can know if the separation is done correctly.

[0003] In the case of a solid separation field, the most direct pH measurement method is to prepare a slice after isoelectric point separation is completed and measure the pH of each fragment. However, it is difficult to measure directly in the case of a liquid separation field. Therefore, in any case, substances with known isoelectric points (isoelectric point markers) are usually separated in the same separation field, and after completion of separation, the calibration curve of pH created using them is used as the standard value in the separation field. A method for determining the pH gradient is used. Patent Document 1 discloses an isoelectric point marker suitable for fluorescence detection isoelectric focusing, and Patent Document 2 discloses an isoelectric marker suitable for ultraviolet absorption detection isoelectric focusing. .

[0004] Even if isoelectric point separation is performed using the same components under the same conditions, the absolute position in the separation field will be different each time. I don't know which position is taking which pH value. In addition, when the liquid separation field is used, a drift phenomenon of the solution occurs, and the solution itself moves substantially in parallel while maintaining the relative pH positional relationship. The pH gradient is measured Can only be judged.

Patent Document 1: JP-A-10-197481

Patent Document 2: Japanese Patent Laid-Open No. 11 23531

Disclosure of the invention

Problems to be solved by the invention

[0005] Normally, isoelectric point separation is performed in one lane, so the sample and the isoelectric point marker are mixed and the isoelectric point is separated. At this time, if the same type of component as the sample component, for example, protein or peptide, is mixed as an isoelectric point marker, it is possible to distinguish the component in the sample from one component at a time. In order to make the distinction easier, if isoelectric point markers are mixed in large quantities, isoelectric point separation may be disturbed due to their influence. Usually, these components are visualized by staining after completion of the separation. In this case, the pH distribution of the separation field cannot be known in real time during the separation process.

[0006] An object of the present invention is to provide a pH gradient determination method capable of accurately determining the pH distribution of a separation field in real time and without performing complicated operations without interfering with the determination of a sample separation result. Is to provide.

Another object of the present invention is that the sample separation result is not determined by the isoelectric point marker, and the isoelectric point of the analyte can be known in real time without requiring a complicated operation. It is to provide an electric point separation method.

Means for solving the problem

[0008] According to the present invention, there is provided a method for determining a pH gradient in a separation field for isoelectric point separation that determines a pH gradient in a separation field for isoelectric point separation including carrier ampholite,

Determine the pH gradient by using carrier ampholite containing multiple fluorescent components with different known isoelectric points and using the fluorescent component as an isoelectric point marker pH gradient of separation field for isoelectric point separation A determination method is provided.

[0009] According to the present invention, there is provided a method for determining a pH gradient in a separation field for isoelectric point separation, which determines a pH gradient in the separation field for isoelectric point separation including carrier ampholite,

Measuring the fluorescence intensity distribution along the separation field using a carrier ampholite containing a plurality of fluorescent components having different known isoelectric points; and A step of determining a pH gradient in the separation field from the position giving the peak of the fluorescence intensity distribution and the known isoelectric point.

A method for determining the pH gradient of a separation field for isoelectric point separation is provided.

[0010] According to the present invention, a step of isoelectrically separating a sample containing a carrier anopholite containing a plurality of fluorescent components having different known isoelectric points and an analyte in the separation field; And measuring the fluorescence intensity distribution;

Determining a pH gradient in the separation field from the position giving the peak of the fluorescence intensity distribution and the known isoelectric point; and

A step of obtaining an isoelectric point of the analyte using the pH gradient

An isoelectric separation method is provided.

The invention's effect

[0011] The present invention provides a method for accurately determining the pH distribution of a separation field without interfering with the determination of the sample separation result, in real time, and without performing complicated operations.

[0012] According to the present invention, the isoelectric point separation is not determined by the isoelectric point marker, and the isoelectric point of the analyte can be known in real time and without requiring a complicated operation. A method is provided.

Brief Description of Drawings

FIG. 1 is a plan view showing a configuration of a microchip that can be used in the present invention.

FIG. 2 shows a calibration curve prepared with a fluorescent isoelectric point marker in Examples.

FIG. 3 shows a fluorescence intensity distribution in a separation channel emitted by carrier ampholite, measured in an example.

Explanation of symbols

[0014] 100 substrates

101 Separation flow path

102 Separation flow path

150 liquid reservoir

151 Liquid reservoir BEST MODE FOR CARRYING OUT THE INVENTION

[0015] The ideal method for determining the pH distribution of the separation field in real time and not interfering with the determination of the separation result of the sample can determine the pH gradient of the separation field without mixing any extra components at all. Is the method. This is because if this can be realized, the original separation pattern of the component to be analyzed can be observed.

[0016] The pH gradient referred to in the present invention means a pH distribution formed along a separation field where isoelectric point separation is performed, that is, in the separation direction of the separation field.

[0017] The minimum mixture for isoelectric point separation is an aqueous solution of the analyte and carrier ampholite. Carrier ampholite is a mixture of compounds having various isoelectric points. By setting acid and alkali electrode solutions on both ends of the separation field filled with carrier ampholite, and applying DC voltage of the anode on the acid side and the negative electrode on the alkali side, each compound contained in the carrier ampholite Converges to the isoelectric point. As a result, a pH gradient is formed in the separation field. The analyte is electrophoresed to a pH position corresponding to its own isoelectric point according to the formed pH gradient and stops.

[0018] If the distribution of the isoelectric point of the carrier ampholite that is always contained in the sample solution and forms the pH gradient of the separation field can be known by some method, other mixed components can be obtained. It is possible to determine the pH gradient of the separation field related to The present inventor has found that there are a plurality of fluorescent components among the component compounds constituting the carrier ampholite. Since these isoelectric points are unchanged, by measuring these fluorescent positions, it is possible to specify multiple absolute pH positions in the separation field, and using a calibration curve created based on them, The pH gradient of the separation field can be determined.

[0019] For example, suppose that the isoelectric point of the fluorescent component of the carrier ampholite is pH 5.0, 7.0 and 9.0. In a system where the separation field pH gradient has been confirmed to be linear, if these three fluorescence positions are measured, at least 5.0 to 9.0 in the separation field based on these positions. The pH gradient of can be determined. For this reason, it is possible to know the pH gradient in the separation field at a certain time even in the case where the pH gradient field once produced in the liquid separation field moves in parallel to the alkali side due to the drift phenomenon. become . Depending on the type of carrier ampholite, the pH gradient may be curved rather than linear. In this case as well, it is possible to know the pH gradient in the separation field at a certain time using the isoelectric point information of the fluorescence component of the carrier ampolite by obtaining the approximate expression of the distribution curve in advance. .

[0020] This method can be performed by utilizing the fluorescence of a commercially available carrier ampholite constituent component. In addition, a carrier ampolite having an isoelectric point marker function can be obtained by imparting fluorescence only to a specific isoelectric point component among the components of the carrier ampholite.

[0021] When detecting proteins and peptides, the ultraviolet light (205 nm to 280 nm) usually used for these detections does not have absorption characteristics, and V and fluorescence characteristics in other wavelength bands. It is preferred to use a carrier ampholite having

[0022] As described above, according to the present invention, when performing normal isoelectric point separation, the pH gradient of the separation field can be determined simply by measuring the fluorescence emitted by the carrier ampholite. You can know the pH gradient of the separation field. Carrier ampholite is the most reliable indicator because it defines the pH gradient of the separation field. Also, since no other markers are mixed, there is no adverse effect on sample separation. Furthermore, it is inexpensive because it does not mix another marker, and it does not require extra time to prepare the introduction solution. As described above, it is possible to provide an accurate, inexpensive, and fast method for determining pH in an isoelectric separation field.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings. In all the drawings, common constituent elements are denoted by the same reference numerals, and description thereof will be omitted as appropriate.

[0024] The method of the present invention can be used in a separation field using a single beam (including a microchip microchannel). Needless to say, it can also be used in other separation fields.

[0025] Here, a case where the method of the present invention is carried out using a fine channel on a microchip will be described. FIG. 1 shows a microchip having a separation channel 101 formed on a substrate 100. Liquid reservoirs 150 and 151 are formed at both ends of the separation channel 101. The shape of the separation channel is not specified as a straight line, and may have a bent portion, for example. Even a microchip having two or more separation channels does not work. For example, the separation channel is set to have a width of 50 111 to 10 mm and a depth of 0.3 m to lmm. With the material of the substrate 100 Thus, for example, a material such as quartz, glass, or plastic is preferably used. The separation channel may have a simple concave structure, or may have a villa or wall-type microstructure for better separation, or may have various coatings applied. . This substrate is covered and sealed with a plate-like substance or a seal made of the same material as or different from the substrate. As the material of the member for sealing and the material of the substrate, a material capable of transmitting the light irradiating the sample entering the fine channel and the fluorescence generated by the sample force, for example, a transparent material can be appropriately selected.

[0026] As a sample for performing isoelectric point separation, for example, an aqueous solution containing an analyte such as protein and carrier ampholite is prepared. Usually, it is possible to use a carrier ampholite aqueous solution of about 0.04 vol% to 4 vol% with 10 _§ to 100; ^ analyte dissolved per separation. In general, gel components may be mixed because the separation results are better for solutions with higher viscosity than water. The preparation liquid (sample) is introduced from the liquid reservoir 150 and filled into the separation channel 101 using capillary action or using external force such as vacuum tweezers. Then, after removing excess preparation liquid remaining in the liquid reservoir 150, the liquid reservoir 150 is filled with an acidic solution, and the liquid reservoir 151 is filled with an alkaline solution. Diluted phosphoric acid or the like can be used as the acidic solution, and diluted sodium hydroxide or the like can be used as the alkaline solution. When an anode electrode is set in the liquid reservoir 150 and a cathode electrode is set in the liquid reservoir 151 and a DC voltage of 100 to 800 volts / cm is applied, the prepared liquid filled in the separation channel 101 starts isoelectric point separation. .

[0027] At this time, an apparatus including a mechanism for exciting the separation channel 101 with light of a specific wavelength and a mechanism for detecting the prepared liquid force and the fluorescence emitted therefrom is prepared. As this apparatus, a system in which an electrophoresis apparatus is set on an electric stage of a fluorescence microscope and a fluorescence intensity measuring apparatus is attached can be used, or a dedicated fluorescence scanner can be used. Examples of excitation light sources include lasers, LEDs, and xenon lamps with filters. Examples of the detection device include a photomultiplier tube (PMT), a photodiode array, and an APD (avalanche photodiode). By using such a fluorescence detection mechanism, it is possible to monitor the fluorescence intensity in the separation channel 101 at each time. For example, a fluorescent microscope equipped with a filter set for fluorescence observation of GFP (Green Fluorescent Protein), which is often used in biological experiments, is used as a fluorescence detection wavelength. A combination of motion stages can be used.

[0028] When a sample prepared using cIEF Ampholyte 3-10 (product code 477491) (product name) manufactured by Beckman Coulter as a carrier ampholite was observed, as the isoelectric point separation proceeded, multiple fluorescent bands were observed. Observed.

[0029] On the other hand, in a sample prepared using the cIEF Ampholyte 3-10 (product code 477491) (trade name), a fluorescent isoelectric marker set (P / N17951; Flu orescent IEF-Marker-Mix manufactured by Fluka) for CE and Gel Electrophoresis) (quotient t¾name) is used as a sample, and the pH gradient is determined from the isoelectric distribution of these markers. Then, when this carrier ampholite is used, the pH gradient in the separation field becomes almost a fountain type. Therefore, once each isoelectric point of the carrier ampholite-derived fluorescent band is determined, a calibration curve related to the pH gradient can be drawn. From this calibration curve, the pH distribution in the separation field can be determined in the pH range that is known to be effective in advance.

[0030] The wavelength of fluorescence transmitted through the filter set for fluorescence observation of GFP is not less than 430 nm and not more than 510 η m, and is different from the ultraviolet light (around 205 η m to 280 nm) usually used for detecting proteins and peptides. is there. Therefore, it is preferable to confuse the fluorescence in this wavelength band with the signal of the analyte for analysis of proteins and peptides. Here, the force S exemplified by GFP as the fluorescence wavelength, and other wavelengths may be used. For example, in the step of measuring the fluorescence intensity distribution, the fluorescence intensity in the wavelength band of 400 nm or more and 780 nm or less can be measured.

[0031] As described above, according to the present invention, there is provided a method for determining a pH gradient in a separation field for isoelectric point separation including a carrier ampholite, and a method for determining a pH gradient in a separation field for isoelectric point separation, which is different from each other. There is a method for determining a pH gradient of a separation field for isoelectric point separation, wherein a carrier gradient containing a plurality of fluorescent components having an isoelectric point is used, and the pH gradient is determined by using the fluorescent component as an isoelectric point marker. Provided. This separation field can be one of the most important.

[0032] Further, according to the present invention, there is provided a method for determining a pH gradient in a separation field for isoelectric point separation including a carrier ampholite, wherein the pH gradient is determined in the separation field for isoelectric point separation. A carrier ampholite containing a plurality of fluorescent components having Measuring the fluorescence intensity distribution along the isoelectric point; and determining the pH gradient in the separation field from the position that gives the peak of the fluorescence intensity distribution and the known isoelectric point A method for determining the pH gradient of a separation field is provided. This separation field force S is the most powerful force S.

[0033] According to the present invention, a step of isoelectric point separation in a separation field of a sample containing a carrier phophorite containing a plurality of fluorescent components having different known isoelectric points and an analyte; along the separation field Measuring the fluorescence intensity distribution; determining the pH gradient in the separation field from the position giving the peak of the fluorescence intensity distribution and the known isoelectric point; and analyzing the pH gradient using the pH gradient There is provided an isoelectric separation method including a step of obtaining an isoelectric point of an object. The analyte can be a protein or peptide. In addition, in the process of measuring the fluorescence intensity distribution, it is necessary to measure the fluorescence intensity in the wavelength band from 400 nm to 780 nm.

Example

[Example 1]

In the following, specific examples will be described by taking the case of the above-described microchip as an example.

[0035] As a microchip, an ICC-DI 05 type (trade name) glass chip manufactured by Micro Chemical Engineering Co., Ltd. was used. This microchip uses borosilicate glass as the material of the substrate 100, produces a linear fine channel on the substrate 100, and further the separation channel 101 becomes a fine channel. It is the microchip which adhered and sealed. This microchip has a force S with two parallel straight channels, and only one can be used at the same time. The actual configuration is the same as in FIG.

[0036] Actually, the microchip was used by attaching a glass reservoir to the liquid reservoirs 150 and 151 of the separation channel 101.

[0037] As the sample solution to be introduced, a mixture of carrier ampholite, a genome for imparting viscosity to the sample, and an isoelectric point marker that emits fluorescence when excited by ultraviolet springs was used. More specifically, Beck Beckman Coulter, Inc. of cIEF Ampholyte 3- 10 (product code 477491) (trade name) 2 volume ^_0/0, manufactured by Beckman Coulter, Inc. of cIEF gel (product code 477497) (trade name: ) 96 volume ⁰ /. Fluorescent isoelectric point marker set (P / N17951; Fluorescent IEF—Marker—Mix for CE and Gel Electrophoresis) (trade name: 2% of commercial products) was used as a sample.

[0038] In order to perform fluorescence observation of the isoelectric point separation, the microchip is placed on an XY electric stage attached to a fluorescence microscope (trade name: AxioPlan 2 Imaging) manufactured by Zeiss, and the microchip is preliminarily provided. Alignment in the XY direction was performed using the alignment mark provided above. In addition, two types of fluorescence filters were set in the fluorescence microscope, and they were exchanged so that fluorescence at different wavelengths could be observed. One of them is for detecting fluorescence emitted by carrier ampholite. No. 09 [Excitation filter (BP450-490), Dichroic mirror (FT510), Noria filter sold for GFP observation] (LP515)] (trade name made by Zeiss). The other fluorescent filter is for observing the fluorescent isoelectric point marker. It is a filter set that is a special combination of three in accordance with the wavelength. [Excitation filter (330WB60), Dichroic mirror (400DCLP), NORIA FILTER (400ALP)] (trade name manufactured by OMEGA OPTICAL).

[0039] Next, an aqueous solution of phosphoric acid (concentration: 0.1 M (concentration unit M represents mol / liter)) is added to the positive side reservoir 150, and sodium hydroxide (concentration) is added to the negative side reservoir 151. : 0 · 02M). Electrodes (not shown) were placed in the liquid reservoir 150 and the liquid reservoir 151, and a DC voltage was applied between the separation channel electrodes. Specifically, for a channel length of about 60 mm, a DC voltage of 3500 V was applied between the liquid reservoirs at both ends.

[0040] Isoelectric point separation was completed about 2 minutes after the start of energization. In the separation channel 101, a pH gradient was created by carrier ampholite, and the five components of the fluorescent isoelectric point marker set formed a convergence band at each isoelectric point. The progress of this isoelectric point separation is as follows: the amount of fluorescence received by the photomultiplier tube (PMT) attached to the fluorescence microscope by stopping energization every 30 seconds and moving the XY motorized stage along the flow path. This was done by plotting.

[0041] First, separation and detection of a fluorescent isoelectric point marker was performed. After the separation is completed, the fluorescence intensity distribution is measured along the separation channel 101, and the peak indicated by each marker component (in each band) Figure 2 is a plot of the distance from the end of the channel (minus side) and the position where the fluorescence is strongest. In this way, the five markers ride on an almost straight calibration curve. Therefore, at least in the range of pH 4.0 to 9.0, the corresponding pH value can be calculated backward using the position in the flow path.

[0042] Next, when the fluorescence intensity distribution along the separation channel 101 is measured by switching to the GFP fluorescence filter, the fluorescence distribution derived from the carrier ampholite is observed.

[0043] However, since it interferes with the fluorescent signal of the fluorescent marker, a complex fluorescent intensity pattern is shown. Therefore, a sample without a fluorescent isoelectric point marker was prepared and the fluorescence intensity pattern was confirmed. Specifically, Beckman Coulter of cIEF Ampho lyte 3 - 10 (product code 477491) (trade name) 2 volume ^_0/0, Beckman Coulter of cIEF gel (product code 477497) (trade name) only 98 vol% The solution mixed with was used as a sample. For this sample, a fluorescence intensity pattern was obtained with the horizontal axis representing the distance from the channel end (minus side) and the vertical axis representing the fluorescence intensity. The distance from the channel end was converted to the isoelectric point (pH) using the correlation shown in FIG. Figure 3 shows the result. Since it was found that each peak was close to the acidic side (pH 4.3-6.2), energization was continued after isoelectric point separation was completed until each peak reached the center of the channel where it was easy to observe. Using the drift phenomenon, the liquid is moved to the alkali side.

[0044] Further, in the experiment using the sample mixed with the fluorescent marker, even if the entire liquid moves in parallel to the alkali side due to the drift phenomenon, the linear relationship of the pH gradient does not collapse. Make sure! /

[0045] As shown in Fig. 3, four characteristic peaks were observed, and these were observed with good reproducibility. The peak that appeared at the acid side end and was pH 2.3 using the calibration curve was not clear, and was not used as a marker. Therefore, three of the four peaks, three peaks of 4.3, 4.6 and 6.2, can be used as absolute internal isoelectric point standards. Therefore, it can be used as an isoelectric point standard in an isoelectric point range of at least 4.3 to 6.2, and is considered to be usable in a wider range in practical use.

[0046] Experiments were actually performed using protein samples. Check protein separation position For this reason, a product stained with the fluorescent dye Cy3 (GE Healthcare Biosciences) was used. As a procedure, first, a sample in which the present protein was mixed with a fluorescent marker was used, and the isoelectric point of the protein was confirmed using the fluorescent marker as an index. Next, with the sample using only this protein, its isoelectric point was measured by the fluorescence of the carrier ampholite, and it was confirmed that almost the same value was obtained. This will be specifically described below.

[0047] A trypsin inhibitor (manufactured by Sigma-Aldrich) was prepared as a protein, and the cysteine residue was labeled with Cy3 and used. 1 · 5 1, 30 mM Tris—HCl (Tris-I) with 2 mM TCEP (Tris 2-chloroethyl phosphate) solution (Invitrogen, Inc., trade name: T—2556) per 10 mg protein solution dissolved in 15 mg / ml Hydrochloric acid buffer solution (ρΗ8 · 0) (manufactured by Nacalai Testa Co., Ltd., trade name: 02435-15) was mixed with 18 · 51 and reacted at 37 ° C for 1 hour. Subsequently, 2 mM Cy3 solution (solvent: DMF (dimethylformamide)) (manufactured by Kanto Chemical Co., Inc., trade name: 10344-07) was mixed with 2 ^ 1 and reacted at 37 ° C for 30 minutes. After removing the excess solution by dialysis after the reaction, a fluorescent protein was obtained. This was prepared as a 1.2 mg / ml protein aqueous solution.

[0048] First, as a sample solution obtained by mixing the protein and a fluorescent marker, cIEF Amp holyte 3- 10 (product codes 477491) (trade name) 2 volume ^_0/0, Beckman Coulter of cIEF Genore (product codes 477,497) (trade name) 94 volume ^_0/0, Fluka Corp. of fluorescent isoelectric point Ma one force a set (P / N17951; fluorescent IEF- Marker- Mix for CE and Gel Electrophoresis) ( trade name) 2% by volume, the protein A solution mixed with 2% by volume of an aqueous solution was used as a sample. In order to observe the state of isoelectric point separation by fluorescence, the above-mentioned microchip is installed on an XY motorized stage attached to a Zeiss fluorescence microscope (trade name: AxioPlan 2 Imaging), and installed on the microchip in advance. Alignment in the vertical direction was performed using the horizontal and vertical alignment marks. In addition, two types of fluorescence filters were set in the fluorescence microscope, and they were exchanged so that fluorescence at different wavelengths could be observed. One of them is to detect the fluorescence emitted by the fluorescent protein, and is a filter set that combines three in particular according to the wavelength. [Excitation filter (ΒΡ525 / 45), Dichroic mirror (FT560) , NORIA FILTER (ΒΡ5 95/60)] (Omega Optical Co., Ltd.). The other fluorescent filter is the fluorescent isoelectric point This is a set of filters that can be used to observe markers and is a combination of three filters according to the wavelength. [Excitation filter (330WB60), Dichroic mirror (400DCLP), Barrier filter (400ALP)] (Omega Optical Co., Ltd.) Product name).

[0049] The positive side reservoir 150 was filled with an aqueous phosphoric acid solution (concentration: 0 · 1M), and the negative side reservoir 151 was filled with sodium hydroxide (concentration: 0.02M). Electrodes (not shown) were placed in the liquid reservoir 150 and the liquid reservoir 151, and a DC voltage was applied between the separation channel electrodes. Specifically, for a channel length of about 60 mm, a DC voltage of 3500 V was applied between the liquid reservoirs at both ends. Isoelectric point separation was completed about 2 minutes after the start of energization. In the separation channel 101, a pH gradient was created by carrier ampholite, and the five components of the fluorescent isoelectric point marker set formed a convergence band at each isoelectric point. Furthermore, the fluorescent protein formed a convergence band at its isoelectric point. The same results as in Fig. 2 were obtained, and a calibration curve in the range of pH 4.0 to 9.0 was created. Next, by switching to a fluorescent filter for fluorescent protein and measuring the fluorescence intensity distribution along the separation channel 101, the fluorescence distribution derived from trypsin inhibitor is observed. The isoelectric point of the trypsin inhibitor estimated from this peak position was 4.6.

[0050] Next, cIEF Ampholyte 3-10 (product code 477491) (product name) 2% by volume, cIEF gel manufactured by Beckman Coulter (product code 477497) (product name) 96 A solution in which 2% by volume of the protein aqueous solution and 2% by volume of the protein aqueous solution were mixed was used as a sample. In order to observe the state of isoelectric point separation with fluorescence, the above-mentioned microchip is installed on an XY motorized stage attached to a Zeiss fluorescence microscope (trade name: AxioPlan 2 Imaging) and installed on the microchip in advance. Then, alignment was performed in the X and Y directions using the alignment mark. In addition, two types of fluorescence filters were set in the fluorescence microscope, and they were exchanged so that fluorescence at different wavelengths could be observed. One of them is for detecting the fluorescence emitted by the carrier ampholite. No. 09 [Excitation Phonoletter (BP450-490), Dichroic mirror (FT510), Noria filter (For GFP observation) LP515)] (trade name made by Zeiss). Another fluorescent filter is [Excitation filter (BP525 / 45), Dichroic mirror (FT560), Barrier filter (BP595 / 60)] (trade name, manufactured by OMEGA OPTICAL). [0051] The positive side reservoir 150 was filled with an aqueous phosphoric acid solution (concentration: 0.1 M), and the negative side reservoir 151 was filled with sodium hydroxide (concentration: 0.02 kg). Electrodes (not shown) were placed in the liquid reservoir 150 and the liquid reservoir 151, and a DC voltage was applied between the separation channel electrodes. Specifically, for a channel length of about 60 mm, a DC voltage of 3500 V was applied between the liquid reservoirs at both ends. Isoelectric point separation was completed about 2 minutes after the start of energization. In the separation channel 101, a pH gradient was created by carrier ampholite, and the fluorescent protein formed a convergence band at its isoelectric point. First, the fluorescence emitted by the carrier ampholite was detected. After the separation was completed, the fluorescence intensity distribution was measured along the separation channel 101, and the same results as in FIG. 3 were obtained. These peaks allowed us to draw a calibration curve from ρ の 4 · 3 to 6 · 2. Next, when the fluorescence intensity distribution along the separation channel 101 was measured by switching to a fluorescent filter for fluorescent protein, a fluorescence distribution derived from trypsin inhibitor was observed. As a result, the isoelectric point of the trypsin inhibitor was estimated to be 4.6. Because of this, this technique can be applied to protein samples and can be applied to peptides as well.

[0052] As described above, according to the present invention, by using a carrier ampholite containing a plurality of fluorescent components having different known isoelectric points, the fluorescent component is used as an isoelectric point marker. The pH gradient can be determined. It is not necessary to specify the compound of the fluorescent component. Carrier ampholite with a pH gradient formed in the separation field! / When a fluorescence intensity distribution is measured along the separation field, carriers whose fluorescence peaks are observed at multiple positions An ampholite may be used. Know the isoelectric points (ρ における) at these multiple positions in advance.

[0053] In order to know the isoelectric point derived from the fluorescent component of the carrier ampholite, it may be as described above. In other words, a sample containing a separately prepared isoelectric point marker and carrier ampholite is subjected to isoelectric point separation, and the pH gradient is determined from the convergence position of the isoelectric point marker in the separation field. The isoelectric point derived from the fluorescent component can be known.

[0054] When such a carrier ampholite is used, the pH gradient can be determined as follows even when actually isoelectrically separating the analyte. That is, the fluorescence intensity distribution is measured along the separation field where the pH gradient is formed, and the fluorescence component of the carrier ampholite is measured. It is also possible to know the pH at a certain position of the separation field, as well as the position that gives the peak of the fluorescence intensity distribution derived from, and the known isoelectric point and force of the fluorescent component. If a plurality of fluorescent components having known isoelectric points with different carrier ampholites are contained, the pH can be known at a plurality of positions in the separation field. Thus, the pH gradient of the separation field can be determined. If the pH at the focusing position of the analyte in the separation field is obtained based on this pH gradient, the pH is the isoelectric point of the analyte.

[0055] Therefore, by using an isoelectric focusing method using a carrier ampholite containing a plurality of fluorescent components having different known isoelectric points, a single isoelectric point is used separately from the carrier ampholite. Without limitation, the pH of the separation field can be determined, and the isoelectric point of the analyte such as protein can be obtained.

Industrial applicability

[0056] The method of the present invention is useful for separating or identifying proteins, peptides, and the like by isoelectric points.

[0057] This application (or claiming priority based on Japanese Patent Application No. 2006-325953 filed on December 1, 2006), the disclosure of which is incorporated herein in its entirety.

Claims

The scope of the claims

[1] A method for determining a pH gradient in a separation field for isoelectric point separation including a carrier ampholite, comprising:

Determine the pH gradient by using carrier ampholite containing multiple fluorescent components with different known isoelectric points and using the fluorescent component as an isoelectric point marker pH gradient of separation field for isoelectric point separation Decision method.

[2] A method for determining a pH gradient in a separation field for isoelectric point separation including carrier ampholite, comprising:

Measuring the fluorescence intensity distribution along the separation field using a carrier ampholite containing a plurality of fluorescent components having different known isoelectric points; and

A step of determining a pH gradient in the separation field from the position giving the peak of the fluorescence intensity distribution and the known isoelectric point.

PH gradient determination method for separation field for isoelectric point separation.

[3] The method according to [1] or [2], wherein the separation field is one of the two.

[4] isoelectric point separation in a separation field of a sample containing a carrier ampholite containing a plurality of fluorescent components having different known isoelectric points and an analyte;

Measuring the fluorescence intensity distribution along the separation field;

A step of obtaining an isoelectric point of the analyte using the pH gradient

An isoelectric separation method comprising:

5. The isoelectric point separation method according to claim 4, wherein the analyte is a protein or a peptide.

6. The isoelectric point separation method according to claim 4 or 5, wherein in the step of measuring the fluorescence intensity distribution, the fluorescence intensity in a wavelength band of 400 nm or more and 780 nm or less is measured.