DE2934479A1 - Electrophoretic analyser for protein and nucleic acid size determn. - with planar transport in first and transverse transport in second gel layer of diminishing pore size - Google Patents

Abstract

An electrophoretic analysis,specially to determine the molecular size of proteins or nucleic acids, is based on a first electrophoretic transport in a first planar carrier layer along its plane. A second transport on an adjacent second layer is in a direction across the plane of the layer. The pore size in the latter is reduced continuously or in steps away from the first layer. A thin planar carrier layer of a gel in homogeneous concn. is in side contact with a thicker gel layer with a pore size hich diminishes outwards. A horizontal louvre grating is fixed by vertical bars which are held together by rubber rings. This simplifies the procedure and speeds it up.

Description

9 'Electrophoretic method for the study of

chemical substances as well as equipment for carrying out this procedure " The invention relates to an electrophoretic method according to the preamble of Claim 1 and a device for performing the method according to the preamble of claim 3.

In research and routine laboratories, the fast and interesting simple investigation of chemical substances, especially the determination of the Molecular size, for example of proteins or nucleic acids.

It is known (see, for example, Slater G.G., Anal.

Biochem. 24 (1968) 215), electrophoresis in polyacrylamide gel, in which the pore size decreases in the direction of transport. These gels are manufactured in vertical glass tubes with a polyacrylamide concentration increasing downwards, i.e. with a gel concentration gradient.

In electrophoresis, for example, the speed slows down of the protein molecules when they reach the pore size in the gel, which corresponds approximately to the molecular size of the proteins. The molecules lay in the gel Due to the concentration gradient, the longer the way back, the smaller it is the molecules are.

The molecules, for example protein molecules, can through Staining be made visible. Proteins with known molecular sizes can be used as reference can be used to determine the molecular size of the proteins under study.

To increase the resolving power is a two-dimensional electrophoretic Process known (see, for example, Margolis J. and Kenrick K.G., Nature 221 (1969) 1056). Here, the substance molecules, e.g. protein molecules, come first separated according to their mobility in a homogeneous gel, which is in a Tube is located.

Then this gel, which has the shape of a narrow strip, becomes brought into contact with the leading edge of a second gel which is in the form of a Has parallelepipeds and is firmly connected to this by polymerisation.

After that, the protein molecules are in the plane by electrophoresis of the second gel transported electrophoretically. The second gel is made of polyacrylamide with pore size decreasing in the direction of transport starting from the starting edge. The time required for the second electrophoresis step is in the The order of magnitude between 18 and 26 hours corresponds to a maximum transport route of about 10 cm in the plane of the second gel layer.

This method is therefore time consuming and also labor intensive because they prepare and manipulate several separate gels, for each Sample a strip-shaped gel for the first dimension and a second gel for each the second dimension.

The object of the present invention is the known method to simplify and shorten.

This task is carried out with the features of the characterizing part of the Claim 1 solved.

The carrier layer materials here are gels such as polyacrylamide, Cellulose acetate, agarose, agarose with essentially linear polymers of polyacrylamide and the like

usable. With the method according to the invention it is possible simultaneously a large number of samples already in the first electrophoresis step in the first relatively thin layer of the carrier medium, e.g. a gel with essentially to separate homogeneous polyacrylamide concentration.

The flat side of this first layer of gel is then in contact with placed on the flat side of a second layer of carrier medium, the thicker can be as the first layer and this completely covers, after which the substances be transported electrophoretically from the first into this second layer. the second layer is made so that the pore size in the transport direction, thus decreases transversely to the plane of the second layer away from the first layer. Since the Transport in the second electrophoresis step only over the thickness of the second If the carrier layer takes place, the maximum transport path is, for example, only 1 cm. The amount of time required to perform the electrophoretic assay is therefore only a fraction of the known method. In addition, the The effort and time required to manipulate separation gels is significantly reduced, there, as mentioned before, for a large number of samples in the first and second electrophoresis step only one carrier gel is required. The resolving power of the invention Method is very good when the concentration gradient of the second support layer is comparatively steep, whereby the molecular zones, e.g.

Protein zones, become appropriately concentrated and sharper.

The thickness of these zones is also only a fraction of that thickness achieved in the previously known methods.

Because of the mentioned concentration effect due to the electrophoretic Transportes across the thickness of the second carrier layer and not in its longitudinal direction there is the further advantage that small amounts of substance can be examined. This means that with the new process a pre-concentration of the substance solutions can be dispensed with prior to electrophoresis. A gradually decreasing pore size the second carrier layer can be obtained by having numerous carrier medium layers builds on top of each other with different pore sizes.

The subclaims contain advantageous developments of the invention Method and in particular also a device for performing the method are specified.

Other features, advantages and uses of the present Invention emerge from the following description of exemplary embodiments based on the accompanying drawing. All of them are described and / or figurative Features shown in any meaningful combination the subject of the present Invention, also regardless of how they are summarized in the claims or their Back-relationship.

It shows: FIG. 1 schematically, in an oblique view, one corresponding to the invention Carrier layer arrangement, in which a thin and a thick gel layer, whose flat sides in close contact with the help of arranged on the respective outside Support grid are held, and Fig. 2 with their facing flat sides in contact standing support layers vertically between two wickerwork, which act as electrodes serve in a trough for buffer solution.

According to FIG. 1, there is a comparatively thin sheet-like carrier layer 1 of a gel of essentially homogeneous concentration is provided, one of which is flat Side in contact with the facing flat side of a comparatively thick one sheet-like carrier layer 2 of a gel with from the contact surface to the outside decreasing pore size. The contact is guaranteed by the support grid 3, which have horizontally running strips slanted in the manner of blinds, which are fixed by vertical rods 4, which are through holes in the horizontal Ledges are led. The rods 4 protrude at the upper and lower edges the carrier layers 1 and 2. Corresponding rods 4 of the two support grids 3 are held together by oval-shaped rings 5. The rings 5 can be made of elastic Material, e.g. rubber, in order to generate the necessary tension. Instead of of the support grid 3, a support network can also be used. The support grid 3 including the horizontal strips and vertical bars 4 are made of rigid Material such as polystyrene or plexiglass; A supporting network can also be made of such material exist.

According to FIG. 2, there is also the comparatively thin carrier layer 1 with a flat side in contact with a flat side of a comparative thick carrier layer 2. Both carrier layers 1, 2 are in a trough that is made of a bottom 6, side walls 7, a rear wall 8 and a front wall, not shown consists. The trough can be made of glass or plastic, e.g. polystyrene or plexiglass exist. The trough is divided into two by the vertically standing carrier layers 1, 2 Subdivided sections, which are almost completely filled with buffer solution and serve accordingly as electrolyte vessels. The support grid 3 according to FIG. 1 can may also be provided, but are shown in the pictorial representation of FIG Omitted for clarity. To ensure that the current is close to 100% flows through the carrier layers, can between the carrier layers and / or the Support grid on the one hand and the part of the trough wall facing each other on the other additional material, e.g. plastic, may be provided.

This is also not shown in FIG. 2. The extra plastic material can either be attached to the trough walls 6, 7, 8 or the support grid 3. on On both sides of the carrier layers 1, 2 there is also a coolant pipe in the trough 12 provided with inlet and outlet ports 11, which in the direction of the arrow from the Coolant is flowing through. The coolant pipe 12 has vertical and horizontal ones Parts. The tube 12 can be cylindrical and is preferably made of glass or thin-walled plastic.

In one plane each parallel to the flat sides of the carrier layers 1, 2 is in each of the electrolyte vessels formed by the trough from horizontal and vertical wires 9 and 10, an electrode formed in a mesh shape is provided.

The wires 9, 10 are preferably made of platinum and are on a Brackets fixed to keep them in the intended plane.

For the preparation of the method according to the invention, for example first a comparatively thick sheet-like carrier layer gel made of polyacrylamide Prepared in a pouring vessel, which consisted of two glass plates, each of the size 11 x 16 x 0.2 cm. Between the glass plates was a rubber strip with a cross section of 1 x 0.5 cm, which ran just inside the glass edges, and the glass plates held at an even distance of 1 cm, provided. The glass plates were clamped together against the rubber strip with the help of clamps. The pouring pot was placed so that the glass plates were vertical.

The three solutions listed in the table below were then used through a hole in the rubber strip filled, so that the solution 1 at the bottom that Solution 2 on top and solution 3 on top.

TABEL Solution buffer solution acrylamide polyacrylamide tetramethylene riboflavin No. Tris- (hydroxymethyl) - (g) (g) (N, N ', N'',N''') - 20 mg / ml in aminomethane-acetic acid ethylene amine (ml) water (ml) 0.08 M, pH 8.6 (ml) 1 48 15 1.1 0.1 0.1 2 49 9 0.4 0.1 0.1 3 49 3 0.24 0.1 0.1 After the casting vessel was slowly placed on a flat side and remained in this position for an hour, the solution components had diffused and formed a continuous concentration gradient of acrylamide which reached from one flat side to the other. The setting of concentration gradients in this way from dissolved substances in water is described, for example, by S. Fredriksson "Isoelectric Focusing in Small Density Gradient Columns", dissertation, Chalmers Institute of Technology, Gothenburg, Sweden, 1975. The casting vessel was then fitted with a daylight fluorescent tube irradiated and thus initiated the polymerization. A polyacrylamide gel backing of the desired thickness formed.

This carrier layer had the highest concentration and accordingly the smallest pores on the flat side below, because this is the highest Concentration of acrylamide before polymerization was.

The first step of electrophoresis was a first thin sheet-like polyacrylamide gel carrier layer of homogeneous concentration in one Horizontal apparatus carried out, for example, in Application Note No. 75, LKB-Produkter, Bromma, Sweden. Serum samples of 10 µl and 0.1 % Concentration of the following standard proteins were used: chicken egg white, human Gamma globulin, hemoglobin and transferrin.

After the initial electrophoresis, the gel backing was thin on cut a size of 10 x 15 x 0.15 cm and its flat side in close contact with the flat side of the thicker gel carrier layer, the manufacture of which was previously described was placed on the side with the lowest polyacrylamide concentration was present. The two gel backing layers were then covered with a support grid in Contact each other held, as shown in Fig. 1, and together with this placed in the trough according to FIG.

The trough was then filled almost to the top with the same buffer solution filled as it was used for the preparation of the thicker gel carrier layer. Thereafter, a direct current of about 1 ampere was applied for two hours at one potential of 36 volts is applied between the two electrodes. Then the support grids were made removed and with a 15 cm long and 0.2 mm thick steel blade with a sharp Edge the gel backing layers cut so that the cut is through a flat side perpendicular to the protein zones. With a method known per se (cf.

e.g., Vesterberg 0. et al, Biochim. Biophys. Acta 491 (1977) 160-166) the protein zones in the gel carrier layers were stained. They appeared as blue Spots on the cut surfaces. When plotting the migration routes of the protein zones in a diagram against the molecular weight results for the standard proteins a linear relationship.

The molecular weight of other proteins can be obtained from such a diagram after their respective migration routes have been determined.

The procedure worked just as well when you put the electrophoresis in The presence of dodecyl sulfate and a polyacrylamide gradient performed. One the advantage here is that the time required for electrophoresis in the second Process step is shortened and is only about 1/3 of the time without dodecyl sulfate, because the protein molecules in the presence of this agent have much higher mobility to have.

Claims (5)

"Electrophoretic method for the study of chemical Substances and device for carrying out this process "Claims: Electrophoretic Process for the investigation of chemical substances, in particular the determination their molecular size, e.g. of proteins or nucleic acids, at which the Substances in aqueous solution initially in a first essentially homogeneous Carrier medium with obstructed convection of the aqueous solution, e.g. in a gel, and then in a second, brought into contact with the first carrier medium, one Carrier medium exhibiting concentration gradients, e.g. in a gel, namely in this are transported electrophoretically in the direction of decreasing pore size, characterized in that the first electrophoretic transport in a first planar Carrier layer (1) essentially in the direction of its layer plane and the second electrophoretic transport in a second sheet-like carrier layer (2) takes place essentially transversely to its layer plane, the second carrier layer (2) runs parallel and with a flat side adjacent to the first carrier layer (1) and a continuous or gradual reduction in pore size towards has away from the first carrier layer (1). 2. Electrophoretic method according to claim 1, characterized in that that the carrier layers (1, 2) only after the first electrophoretic transport be brought into contact with each other. 3. Apparatus for performing the method according to claim 1 or 2, characterized characterized in that the second carrier layer (2) is thicker than the first carrier layer (1) is. 4. Apparatus according to claim 3, characterized in that the two carrier layers (1, 2) in the form of separate plates or applied to separate plates are. 5. Apparatus according to claim 3 or 4, characterized in that the carrier layers (1, 2) by means of support plates, grids or networks (3, 4, 5) during the second electrophoretic transport lying flat against each other in a trough (6, 7, 8) can be positioned with buffer solution.