JPH07506421A - Electroelution method of gel containing separated proteins or charged macromolecules such as DNA/RNA, and apparatus and means used therefor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] name of invention containing separated proteins or charged macromolecules such as DNA/RNA. Electroelution method for gels and equipment and means used therefor Missing sake 1 The present invention relates to isolated proteins or charged macromolecules such as DNA/RNA. A method of the type indicated in the preamble of claim 1 for electroelution of gels containing Regarding. The method of the invention is novel in the "characterizing" part of claim 1. . Furthermore, the invention relates to devices and means for use in this method.

11 Long Yong Proteins or DNA/RNA that differ in size and/or charge amount Gel electrophoresis is widely used for separation. These compounds have different migration distances. Because of this, it is difficult to stain gels or transfer to membranes for antigen-antibody experiments. and other affinity interactions. however, Traditionally, to recover individual protein bands, gels are typically This segment is divided into segments containing the The difficult and time-consuming method was to homogenize and elute the pigment.

Moreover, this method is inefficient and yields only a very dilute product. There are many bad things.

U.S. Pat. No. 3,956,099 discloses that during the electrophoretic operation A complex system suitable for continuous operation is disclosed, which separates the machine into individual rooms. death However, this method requires prior separation of macromolecules in a conventional gel before elution. (Using separation equipment for large-scale purification of proteins.

U.S. Patent Nos. 4.049.534, 4.725.348 and No. 4.747.918 describes small gel fragments containing a single component. Disclosed are methods and components suitable for electroelution. In this method, the desired The process of localizing and ablating the molecule is a time-consuming and inaccurate operation. Therefore, all components separated in the gel should not be This is not an appropriate way to consider it.

EP 0 380 313 specifically describes the transfer of high molecular weight compounds. Field inversion electric absorption transition (field 1nversion ele) suitable for ctroblotting) and elution apparatus are disclosed. Even with this method, Since the desired band must be identified, the above U.S. patent no. 4.049.534, 4.725.348 and 4,747.918 It has similar limitations as the method disclosed in the specification.

U.S. Pat. No. 4,181,594 states that slab gel contains Matrix recovery device where compounds are electroeluted directly into the upper wells is disclosed. Each compound consists of several parts separated by walls of considerable thickness. Elute into wells. Movement becomes uneven or inefficient due to the thickness of the wall. there is a possibility. The wells are filled with different protein indicators. this device does not have a dialysis membrane, so the resulting product contains salts from the gel and surfactant (det agents).

Blotelutor (registered trademark) device us manufactured by Biometra, Gotting en, Germany) is a specialized device for eluating proteins from two-dimensional gels. It is. Proteins are eluted into wells separated by walls with considerable thickness. do. In this case as well, movement may be uneven due to the thickness of the wall. moreover, The method of this device does not use physiological buffer and requires equilibration as a pretreatment. Therefore, there is no step to ensure that the gel position is fixed during elution.

In any of these conventional systems, all the chemical reactions in the -dimensional gel are None meet the traditional requirements of separating and coeluting compounds. these If you have a unique system, you can biologicalize complex protein/DNA mixtures. This will greatly advance scientific screening methods.

Moxibustion Therefore, the object of the present invention is to analyze large molecules such as proteins or DNA/RNA. The purpose is to make it possible to separate children in advance. In the method of the present invention, protein Charged macromolecules such as They can be separated throughout and eluted simultaneously. By doing this, you can A compound is a single compound or a narrow range of flags that includes multiple compounds in close proximity. The large amount of sodium dodecyl sulfate (SDS) coexisting at the same time is divided into parts are removed.

In the present invention, it is novel that the entire gel is eluted all at once. aforementioned From what I have learned, conventional methods generally require gel matrix treatment at the end of electrophoresis. The basic method is to cut the wood directly. After homogenization, the material is electroeluted move it to As mentioned above, in particular, the entire protein content of the gel is lumped together ( Typically, it is desirable to screen 20-30 fractions at the same time. In certain cases, such methods are very difficult and time consuming.

The present invention further provides that the method of the present invention can be used to separate and synchronize all compounds in the entire gel. Provides an electric elution device that elutes at times. This device is very efficient in converting ordinary gels into (0.5-1 mm thick), but very thick (3-4 mm) gel elutes. Also suitable for

Hereinafter, the method and apparatus of the present invention will be explained with reference to the drawings.

・Nano tongue■ FIG. 1 is a schematic diagram of a multi-chamber electroelution device of the present invention.

FIG. 2 is a photograph of a gel eluted from the apparatus shown in FIG. 1 by the method of the present invention.

Figure 3 shows the fractionation of a complex mixture of tannoliths by the electroelution device shown in Figure 1. It's a photo.

Figure 4 shows cells for fractions eluted from a multi-chamber electroelution device. It is a graph showing the reaction.

Figure 5 shows that the fractions eluted from the multi-chamber electroelution device are non-toxic. It is a graph showing.

Details of Kaya day FIG. 1 shows a cross section of the device. This whole electric elution device consists of graphite electrode and its terminal Made of plastic that is resistant to surfactants.

The device consists of a support (1) containing a graphite anode (4) and a sponge filled with a buffer solution ( 5) It consists of. The support (1) has a reservoir, and when the frame (2) is mounted The sponge is pushed out (receiving the excess buffer solution) on the sponge. has a semi-permeable membrane such as a dialysis membrane, and the frame is firmly attached to the support using a screw that is turned with your fingers. It is continued.

The dialysis membrane may optionally be fixed to the sponge, for example by gluing. graphite The cathode (3) is embedded in the safety cover (8).

The frame has triangular number chambers (7) lined up in parallel, and the surface of the chamber is transparent below. The chamber is sealed by close contact with the surface of the deposited film (6).

The capacity of each chamber is 15 m1 or less, preferably 1. < is approximately 3 ml.

There is a depression in the frame, and the gel (9) is placed in it.

A filter paper (10) moistened with buffer solution is sandwiched between the gel (9) and the graphite cathode (3). is preferred. One of the parts of the device has gel removed precisely according to the size of the depression. There is a mold.

The mold is made of clear acrylic plastic and the location of each well is clearly marked. It is. The mold also has a stopper adjustment to accurately position the mold before cutting. A line is drawn.

A voltage potential of approximately 40 volts is applied. The power supply must also be adjusted to be able to apply this volt. It must have a regulating function. Power is provided by wires (11 and 12 respectively) and connect to the cathode (3) and anode (4). The power supply is embedded in the safety cover (8). Also good.

The time required to complete elution is generally 8 minutes to 1 hour for a 0.75 mm gel. It's 5 minutes. Once the elution is complete, reverse the direction of the current for 10 seconds to ensure that it sticks to the membrane. The product is easily removed from the plastic material through the collection port on the side of the device. Collect with a cupipette.

In the electroelution method, first, as a pretreatment, the gel containing the molecules to be separated is soaked in a buffer solution. Begin by equilibrating for about 30-40 minutes, preferably for about 30-40 minutes. for example, If the product is used for cellular analysis, the preferred buffer is a low ionic strength physiological buffer. A buffer solution, more preferably a 2mM IJ salt buffer (pH 6.5). equilibrium During the incubation period, the gel swells to its maximum size, thus limiting the elution. The position of the gel can be reliably fixed throughout the process. Change the buffer three times and Remove excess salt and ionic detergents from the Remove the gel from the buffer, place it on a clean glass plate, and elute. The part to be removed is cut out using a mold. The preferred method of use is to use pre-stained molecular weight Draw a line around the gel with a marker. By aligning the mold adjustment line with the marker, This allows for accurate positioning of the gel and reproducible resection. You can also do it. Although it is important to parallelize the gel bands and the elution chamber, this method This can be done reliably.

The sponge of the device support is filled with a buffer solution. Sponge the dialysis membrane soaked in buffer solution and attach the frame with the thumbscrew. The sponge acts as a support for the membrane. When the sponge is pressed, each chamber is sealed. Fill the chamber with buffer solution and place the mold. to move the gel into the recess of the frame. Two thick filter papers that match the size of the gel. Soak the sheet in buffer and place it on top of the gel. Equipped with a graphite cathode, preferably about 40 volts. Apply the root volt potential. During elution, charged molecules fill the chambers from the gel. Transfer to buffer. The upper part of the wall separating the triangular chambers is sharply pointed, making it ideal for There are as few surfaces as possible that impede the movement of objects. Ionic surfactant (SDS ) is effectively removed in the buffer solution after passing through the dialysis membrane and moving towards the anode. Ru. The time required to complete elution depends on the compound being studied and the matrix used. Although the time varies accordingly, it is preferably 8 minutes to 16 minutes. After the elution has finished, for a short time, Preferably, the direction of the current is reversed for about 10 seconds to easily peel off the molecules attached to the membrane. to save. After elution, apply a pipette to the opening on the side of the frame and suck up the product. to recover. After collecting the product, disassemble the device and add protein/DNA to the gel. Apply staining to confirm completion of migration. Enough PBS for all fractions The product is used for cellular analysis while being made isotonic with the addition of XIO.

A second embodiment of the invention uses this device to simultaneously perform electroelution and electroabsorption transitions. This is something that should be carried out from time to time.

In this embodiment, one suitable membrane is inserted into one side of the device and the changing parts of all chambers are Cover the minutes. This allows the molecules to move into the solution and be absorbed and transferred to the membrane at the same time. Ru. In a preferred form, the T cell response to the protein mixture being separated sex and antibody responses can be analyzed simultaneously, easily and accurately.

A third embodiment of the invention is a simple and rapid method of purifying a single compound. Ru. This method involves gel strips or electroabsorption-transferred substances that are being separated. The process of removing the membrane and staining or reacting with specific probes The method consists of the step of localizing the desired compound. In a preferred embodiment In some cases, the probe is a monoclonal antibody. Each elution chamber is sharply pointed. It is shown on the membrane by the influence from the wall of the chamber. When staining gel pieces, elution Numerous 2-3 mm holes are drilled indicating the localization of the chambers. Accurately identify each elution chamber Marking molds facilitate accurate drilling. these The sample allows for accurate identification of the chamber containing the compound of interest.

As seen in Figure 2, the method and apparatus of the present invention effectively dissolve all parts of the gel. can be released. The gel is divided into two parts, A and B.

Part A of the gel was removed before elution and stained for protein. .

The elution gel (part B) is used to confirm the completion of protein transfer after the elution step. It has been dyed for sex.

In the gel, there is a thin line (light line) indicating the position of the edge of the chamber wall, and two vague marks at the bottom of the gel. I can see parts. These two findings highlight two important points: .

l: By making the chamber walls sharp, there are as few surfaces as possible that impede optimal movement. Ru.

2: Air bubbles at the bottom of the gel interfere with protein movement.

The photograph in Figure 3 shows the fractionation of a complex mixture of proteins.

The filtrate of Mycobacterium tuberculosis culture was subjected to 10-20% 5DS-polyacrylamide electrophoresis ( 10-20% 5DS-PAGE) and the multi-chambered electrolyte solution of the present invention. It is fractionated using an extraction device. The filtrate and the obtained fraction were subjected to 5DS-PAG Analyzed by E and silver staining.

Lane F is the filtrate of Mycobacterium tuberculosis.

Lanes 1-18 are fractions 1-18.

Figure 4 shows the proliferation response of splenic lymph cells collected from mice infected with Mycobacterium tuberculosis. It shows. A 1:5 dilution of a fraction of Mycobacterium tuberculosis culture filtrate. Cells were grown in stimulated culture medium. The growth response to the intact filtrate was 1860 0 cpm, and the response of unstimulated cells was 500 cpm. Measurement of proliferation , a liquid scintillation meter that incorporates 3■thymidine into the DNA of proliferating cells. Measured by number.

From Figure 5, it can be seen that the fraction obtained by the multi-chamber electroelution device of the present invention is toxic. It can be seen that there is no

Mild stimulation of multi-chamber electroelution device fractions, moderate 1:5 to 1:500 stimulation. It was diluted within a range and examined. A clear dose-dependent relationship for all fractions, i.e. It was observed that the higher the dose, the greater the proliferative response. this Their results highlight the non-toxicity of the products of electroelution devices.

As is clear from the foregoing, the method and apparatus of the present invention have the following major advantages: I understand.

First, all the components in a one-dimensional gel, which is commonly used on a daily basis, are separated. It is possible to quickly and easily carry out simultaneous elution operations. moreover , it becomes possible to use one-dimensional gels with various thicknesses, and therefore the present invention has maximum adaptability. For this reason, the electroelution device can be used to Can be part of a purification process and analyze the product quantitatively and qualitatively You can also do it.

Triangular wells allow all protein bands to migrate evenly Can be done.

The separation frame can be extended towards the sponge to create an efficient container that is easy to handle. I can do it.

The buffer is non-toxic (and free of chloride ions, which can be a problem during elution). stomach. The same buffer is used in all parts of the electroelution device, saving labor.

Once elution is complete, the product is sucked out, so there is no need to disassemble the machine. to this Therefore, high purity can be maintained. The product is completely non-toxic and can be used for many different purposes. can be used.

Finally, templates can be used to perform accurate and reproducible fractionation. .

F 1 2 3 4 5 6 7 B 910111213141516171 11cpm x 1000 Fraction (pmx 1000 international search report international search report NO, PCT/DK 92100058 Front page continuation (51) Int, C1, 6 identification symbol Internal reference number GOIN 33/483 F 7055-2J33158 A 7055-2J I

Claims (12)

[Claims] 1. Contains charged macromolecules such as isolated proteins or DNA/RNA. Electroelution methods for gels such as polyacrylamide or agarose gels with After equilibration of the gel as a pre-treatment, electrolysis using an appropriate specific buffer is performed. The gel is separated in advance by eluating the entire gel at once in the extraction device, and a large A large molecule can be divided into a narrow range of molecules containing a single or several closely located compounds. and remove most of the sodium dodecyl sulfate (SDS) used. However, prior to this, the template allows for accurate and reproducible fractionation. An elution method characterized by cutting the gel. 2. An elution method according to claim 1, comprising: (a) using an excess of salt; As a pre-treatment, preferably for about 30-40 minutes to remove most of the SDS. equilibrating the gel in a buffer solution; (b) using a specific buffer solution with low ionic strength in the elution step; (c) Reverse the direction of the current for a short period of time, preferably about 10 seconds, to A process of making the particles easy to peel off, and (d) Add sufficient amount of PBS x 10 to all fractions to divide the fractions. The process of making it isotonic An elution method comprising two or more of the following. 3. In the elution method according to claim 2, the buffer used in step (b) is phosphoric acid. An elution method characterized in that the elution method is a salt, preferably at a concentration of 2mM. 4. All the components separated in the gel according to claim 1 are separated in a large number of In an electric elution device that simultaneously elutes a) a support (1) containing a graphite anode (4) and a sponge covered with a dialysis membrane (6); (5) (However, the dialysis membrane may optionally be fixed on the sponge), b) a plurality of parallel chambers (7) each bounded at the bottom by said dialysis membrane; c) an opening on the side of said frame to collect the isolated compound; mouth, d) a graphite cathode (3) embedded in the safety cover (8); and e) A dissolution method characterized by a mold that performs electroelution after precisely excising the gel. Output device. 5. The elution device according to claim 4, wherein said chamber (7) has an acutely pointed tip. Because of its triangular shape, all the compounds being separated in the gel have optimal migration. An elution device characterized in that it is capable of: 6. The elution device according to claim 4, wherein the dialysis membrane (6) concentrates the desired compound. The salts and ionic surfactants are removed after passing through the dialysis membrane. elution device. 7. The elution device according to claim 4, wherein the spout is covered with a dialysis membrane (6). (5) is forced into close contact with the frame (2) to form a number of closed cells. An elution device characterized by: 8. The elution device according to claim 4, wherein the power source is embedded in the safety cover (8). an elution device in which the power source is capable of automatically reversing the direction of the current. 9. In the elution device according to claim 4, the opening on the side of the device can be approached from the outside. The resulting fraction can be collected without dismantling the device. It is characterized by the fact that it is safe to handle, and that it is possible to obtain a product of high purity. Elution device. 10. The elution device according to claim 4, wherein an adjustment line is written on the mold. This makes it possible to accurately and reproducibly position the gel as molecules move. An elution device that is characterized by the ability to 11. In the elution device according to claim 4, elution and absorption transfer can be performed simultaneously. An elution device characterized in that a membrane is inserted into the elution device. 12. In the mold used in the elution device according to claim 4, the position of each chamber is clearly indicated. An elution device characterized in that the chamber containing the desired single molecule can be identified.