Electro-Blotting of Electrophoretically Resolved Fluroescent-Labeled Saccharides and Detection of Active Structures With Protein Probes

This application is related in part to two other co-pending U.S. applications filed concurrently with the present application on Feb. 16, 1990. One related application is Ser. No. 07/481,361, which application is entitled "Two-Dimensional Electrophoretic Separation of Carbohydrates" invented by Brian K. Brandley, a co-inventor of the present invention; the other application is Ser. No. 07/483,043, which is entitled "Fluorescent Tag for Sugar Electrophoresis" also invented by Brian K. Brandley and Michael Tiemeyer, who are co-inventors working in the same research organization as the present inventors with an obligation to assign the invention to the same entity. The above-referenced applications are each incorporated herein by reference in their entireties.

This invention relates generally to separation techniques such as electrophoresis and electro-blotting which allow for the separation of particular materials and then testing of those materials for activity utilizing labeled probes. More particularly, the invention relates to utilizing electrophoresis followed by electro-blotting in order to separate mixtures of saccharides containing very small amounts of fluorescent labeled saccharides and then determining the activity of such saccharides by using labeled protein probes.

Electrophoresis is a well known technique for the separation of a charged species by utilizing their differences in rate of migration under the influence of an electrical field. The procedure has proved invaluable for the resolution and isolation of complex biological substances such as enzymes, serums, carbohydrates, proteins, DNA and RNA. Most analytical electrophoresis methods are based on zone-electrophoresis in which a thin zone of a sample material is applied to the electrophoretic medium. The electrophoretic migration of the sample components results in the formation of fractional zones. These zones can be examined and studied by applications of standard electrophoretic practice such as fixing, staining and washing to remove buffers. Desirably, the electrophoretic media is a thin gel film coated on a suitable support, commonly glass or plastic. Such an arrangement permits the electrophoretic separation to be achieved in a minimum of time with a maximum degree of resolution.

Various hydrophilic colloids, for example, starch, agarose and cellulose derivatives have been used in forming electrophoretic gel films, but polyacrylamide is preferred. One reason for preferring polyacrylamide is that gels can be prepared from it having a wide range of pore size. This is accomplished primarily by varying the ratio of acrylamide polymer to the N, N', methylenebisacrylamide cross-linking reagent.

The resulting polyacrylamide gels provide high resolution electrophoretic separation of important biopolymers, for example, proteins and nucleic acids. In addition, the absence of ionized groups in polyacrylamide gels render such gels suitable as an anticonvection medium for isoelectric focusing.

Once the electrophoretic techniques have been applied in order to separate the materials in the gel, it is necessary to transfer the separated materials from the gel to a support where they can be tested. A number of procedures are available for transferring the electrophoretically resolved materials from the gel. One such procedure involves electro-blotting. This type of transfer procedure involves transferring the resolved bands within the gel to a support matrix such as a nitrocellulose sheet. The transfer is carried out by the application of an electric field and therefore is distinguishable from a more conventional alternative which involves the capillary transfer of such materials usually used in techniques such as southern and northern blotting.

In accordance with the present invention mixtures of saccharides are resolved into groups and tested for their affinity to particular proteins. The process is carried out by conjugating the saccharides in the mixture to a charged molecule and then subjecting the charged conjugates to electrophoretic techniques in order to separate the different saccharides or groups of saccharides from each other. After carrying out electrophoretic resolution, the resolved bands within the gel are electro-blotted onto a membrane which is preferably a charged nylon membrane. The nylon membrane provides a stable record of the electrophoretic separation in the gel. The membrane having the bands of saccharides thereon is then subjected to testing techniques with various labeled protein compounds. If the protein binds to the saccharide material, the binding can be detected by the probe and the saccharide is determined as being one of particular interest due to its binding affinity to the protein and can be further studied.

A primary object of the present invention is to provide a method for separating mixtures of saccharides into closely related groups of saccharides and determining which groups of specific saccharides have particular binding affinities to particular proteins.

An advantage of the present invention is that large numbers of different saccharides each present in small amounts in mixtures can be readily separated and readily tested for their affinity to large numbers of proteins.

A feature of the present invention is that it involves a unique combination of separation and probing techniques which can be readily and efficiently carried out to obtain large amounts of information with respect to particular saccharides.

These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the structure, synthesis and usage as more fully set forth below, reference being made to the accompanying general structural formulae forming a part hereof.

Before the present method for separating and testing saccharides is described, it is to be understood that this invention is not limited to the particular saccharides, proteins or process steps described as such compounds and steps may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intending to be limiting since the scope of the present invention will be limited only by the appended claims.

It must be noted that as used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes mixtures of proteins, reference to "an oligosaccharide" or a saccharide includes reference to mixtures of oligosaccharides or saccharides, and reference to "the electrophoretic processing step" includes a variety of similar steps of the type described herein.

The first step of the present invention involves binding the saccharide molecules to be tested to a charged molecule. This is done in order to allow for the mixtures of saccharides to be separated from each other by electrophoretic techniques which require that the materials being separated have electrical charges thereon. In connection with the present invention, the saccharides are conjugated with 4-amino-1-naphthalene sulfonic acid (hereinafter ANSA).

The 1-amino-4-naphthalene sulfonic acid (ANSA) used in connection with the present invention has the following structure: ##STR1## The -NH.sub.2 amino group is indicated as being at the "1" position and the -SO.sub.3.sup.= group is at the "4" position. The shared double bonds in each ring structures provides the fluorescent character to the ANSA when the ANSA is exposed to U.V. light.

Depending on the type of saccharide in the mixture being resolved, there may be additional -SO.sub.3.sup.= groups attached to the basic ring structure. The additional -SO.sub.3.sup.= groups provide additional charge to neutral saccharides. If two -SO.sub.3.sup.= groups are present, the molecule is referred to as ANDA and if three are present ANTS, for example, (8-aminonaphthalene-1,3,6 trisulfonic acid).

After the saccharide/ANSA conjugates are formed, they are subjected to standard electrophoretic techniques in order to resolve the different saccharides from each other. The electrophoretic resolution provides a gel wherein the saccharides are separated from each other in different bands along the length of the gel.

The separation techniques utilized in connection with the present invention have been found to work particularly well in connection with smaller saccharides. More specifically, the gel electrophoresis has been found to be particularly useful in separating mixtures of monosaccharides, disaccharides, and trisaccharides. Conventional procedures are generally not capable of providing sufficient resolution to separate away smaller saccharides into distinct bands. The addition of the ANSA group provides a sufficient amount of charge to allow for the separation of the smaller saccharides into distinct groups but does not apply too much charge so that the charge quality overwhelms any other quality of the saccharides and thus does not provide for resolution among different types of closely related saccharides. Further, the shared double bonds within the ring structures of the ANSA provide for the fluorescent capability of the conjugates formed. Accordingly, when different bands of saccharides are separated away from each other, it is possible to visually view these bands simply by the application of ultraviolet light.

It is pointed out that some mono- and disaccharides can only be resolved into distinct groups via electrophoresis by using ANSA. The monosaccharides include glucuronic acid, iduronic acid and galacfuronic acids. The disaccharides include:

2-acetamido-2-deoxy-3-0 (BetaD-gluco-4-ene pyranosyluronic acid)-D-galactose;

2-acetamido-2-deoxy-3-0 (BetaD-gluco-4-ene pyranosyluronic acid)-4-0-sulfo-D-galactose; and

2-acetamido-2-deoxy-3-0 (BetaD-gluco-4-ene pyranosyluronic acid)-6-0-sulfo-D-galactose

The use of such a fluorescent tag provides a number of advantages over and above the use of other types of tags. For example, a fluorescent tag is substantially safer and less expensive than the use of a radiolabel. Further, the use of a fluorescent tag is substantially less cumbersome and more efficient than the use of antibody-linked enzyme tags. These advantages are obtained concurrently with the overall advantage of providing a tag which allows for greatly improved resolution especially as used in connection with smaller saccharide compounds.

The separated bands of conjugates within the gel are then transferred to the surface of a membrane. A number of different types of membrane surfaces can be utilized in connection with the invention. However, nylon is preferable. The transfer of the saccharide/ANSA conjugates from the gel to the surface of the substrate is carried out by utilizing electro-blotting techniques. The electro-blotting is carried out for a sufficient period of time to allow substantial amounts of the conjugates within the gel to transfer to and bind to the surface of the substrate thus providing a permanent record of the separated bands of conjugates on the surface of the membrane.

The electro-blotting procedures which can be used in connection with the present invention are procedures which are generally known to those skilled in the art. In general, a gel having the separated conjugates thereon is placed in contact with a membrane surface. The membrane surface which is preferably a charged nylon surface is preferably first wetted with a buffer in which the electro-blotting procedure will be carried out. The membrane surface may be coated with polyisobutylene methylmethacrylate. What is arbitrarily chosen as the cathode side of the gel (i.e., ultimately towards the negative electrode when positioned in the electro-blotting tank) is placed in contact with the surface of the nylon substrate after the substrate has been moistened with the electroblotting buffer. Any air bubbles between the gel and the nylon membrane should be removed by gently pushing the nylon substrate against the gel using powder-free gloved fingers. A piece of nitrocellulose can be placed on the opposite side of the gel and all of the air bubbles should construct is then placed in the electro-blotting tank which contains a buffer solution and has an anode and a cathode therein. The power supply is then turned on and the power supply will draw the electrically charged saccharide/ANSA conjugates out of the gel and onto the charged surface of the nylon substrate. The transfer time is dependent somewhat on the thickness of the gel and the size of the conjugates being transferred to the nylon substrate. The transfer can be monitored by viewing the transfer under U.V. light to insure complete transfer of all of the materials to the nylon substrate surface. Overnight transfer is reliable and convenient.

One of the surprising discoveries of the present invention is that the specific bands of conjugates in the gel are even more clearly resolved and distinguishable from each other when the transfer is made to the nylon substrate surface. While not wishing to be bound to any particularly theory, it is believed that greater resolution is obtained on the nylon surface because of the diffusion of light in the gel when the conjugates are exposed to U.V. light. Regardless of the reason, it has been found that distinct, separate bands of conjugates are formed on the nylon substrate surface.

After the conjugates on the membrane surface have been secured to the surface, the specific saccharides within each of the visually detectable groups can then be tested for their affinity to other molecules. This testing is generally done by first forming conjugates of molecules to be tested by binding such molecules to a label. For example, protein molecules are bound to a radiolabel. The conjugates of radiolabeled proteins are then brought into contact with the saccharide/ANSA conjugates on the surface of the membrane. If the proteins have an affinity to the saccharides on the membrane, they will bind to the saccharides, thus forming double conjugates, i.e., the saccharide/ANSA conjugates bind to the protein/label conjugates.

After the protein/label conjugates have been allowed to remain in contact with the membrane surface for a sufficient period of time to allow for complete binding, the membranes are washed thoroughly in order to remove any unbound protein. After the unbound proteins are removed, the bound proteins, if any, are detected by utilizing the label attached to the proteins by procedures such as radiography.

The separation methodology of the present invention can be utilized in order to test a variety of different types of compounds for their affinity to the saccharides on the nylon substrate. For example, the invention can be utilized in order to test the affinity of certain lectins for their affinity to the saccharides. Particular types of antireceptor proteins known to be positioned on viruses and to be attachable to certain saccharides on cell surfaces can be tested. Further, the affinity of certain growth factor proteins can be tested. It is believed that the attachment of certain saccharides to growth factor proteins can effect the activity of the growth factor protein in vivo.

The molecules to be tested, such as the protein molecules to be tested for their affinity to saccharides, must, of course, be bound to a label which is later detectable. A variety of different types of labels known to those skilled in the art can, of course, be used. For example, it is possible to utilize radiolabels which are later detected by the use of autoradiography. It is also possible to attach the protein molecules to an antibody which itself is bound to an enzyme such as horseradish peroxidase which can be detected by the addition of reagents which cause a color change. Procedures for attaching the labels to the proteins or other molecules to be assayed are well known to those skilled in the art.

The following examples are provided so as to give those of ordinary skill in the art a complete disclosure and description of how to carry out the processes of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviation should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.

Charged saccharides in the form of heparin fragments from partial nitrous acid digests are reacted with ANSA to form conjugates. The reaction is carried out in 10 to 100 mM sodium acetate buffer (pH 5.0). The heparin fragments are present in an amount of 0.01-1 umol/ml and are reacted with ANSA and sodium cyanoborohydride in a ten fold molar excess with respect to the reducing end sugar of the heparin fragment. The derivatized heparin fragments are then subjected to electrophoretic resolution in 40% acrylamide/5% bis gels, with a Tris/ glycine buffer system (25 mM Tris, 195 mM glycine, pH 8.3). The electrophoretic gel is to be run at 300 volts for approximately 90 minutes. The gels are to be immediately electro-blotted using a Biorad apparatus onto a Zetaprobe membrane (of the type commercially sold by Biorad). The electro-blotting is carried out utilizing standard techniques and 100 volts for one hour with the same Tris/glycine buffer system. After completing the electro-blotting, the Zetaprobe membranes are removed and air dried.

The drying membranes have the separated heparin fragments bound thereon. These membranes with the heparin bound thereon are stable on PBS at least overnight. Accordingly, such blots can be blocked with 2% PVP40 (Sigma) in PBS for 1 hour at room temperature and then probed with proteins (radioiodinated bFGF) overnight at 4.degree. C., in PBS plus 2% PVP40. After allowing any binding to take place, the membrane are washed with the same buffer three times, and then dried. Bound protein can be detected by autoradiography. The ANSA provides a fluorescent label on the heparin fragment will allow for the direct visualization of the blot and comparison of it with the autorad, i.e., the membrane having the radiolabeled proteins bound thereto.

Neutral saccharides in the form of tetrose derived from asialo-GM1 were charged by reacting them with ANSA to form conjugates. The reaction conditions were 10kb/100 mM sodium acetate buffer (pH 5.0), asialo-GM1 at 0.01-1 uM/ml, ANSA and sodium cyanoborohydride in tenfold molar excess to the reducing end of the sugar. Derivatized saccharides were resolved in 40% acrylamide/5% bis gels with a Tris/glycine buffer system (25 mM Tris, 195 mM glycine pH 8.3) run at 300 volts for approximately 90 minutes. The gels were immediately electro-blotted using a Biorad apparatus onto Zetaprobe membranes (manufactured by Biorad) by standard techniques (100 volts, 1 hour) with the same Tris/glycine buffer system.

It was found that the ANSA tag did not apparently impart sufficient charge to keep these neutral saccharides stably associated with the membrane in buffer solutions. Accordingly, the membranes were first wetted with hexanes, then soaked in 0.1 percent polyisobutylene methylmethacrylate in hexanes for 45-60 seconds and were dried. These membranes were probed in 50 mM buffers.

The instant invention is shown and described herein in what is considered to be the most practical, and preferred embodiments. It is recognized, however, that departures may be made therefrom which are in the scope of the invention and that obvious modifications will occur to one skilled in the art upon reading this disclosure.