WO1994012691A1

WO1994012691A1 - USE OF FLUORESCENT pH-MARKERS IN MOVING BOUNDARY GEL ELECTROPHORESIS SYSTEMS

Info

Publication number: WO1994012691A1
Application number: PCT/US1993/010414
Authority: WO
Inventors: Erich Gombocz; Eva Cortez; David Rammler
Original assignee: Labintelligence, Inc.
Priority date: 1992-12-02
Filing date: 1993-10-29
Publication date: 1994-06-09

Abstract

Gel electrophoresis methods and compositions are provided, employing a fluorescent pH marker which allows for monitoring of the progress of the gel, and identifying the migration boundary and the pH at particular sites in the gel. By using pH gradients, particularly transverse gradients, pH-related migratory characteristic can be identified, so as to provide for a distinctive characteristic for a sample component and improve the opportunities to separate sample components which might migrate together at a particular pH.

Description

USE OF FLUORESCENT pH-MAR ERS IN MOVING BOUNDARY GEL ELECTROPHORESIS SYSTEMS

INTRODUCTION Technical Field

The field of this invention is gel electrophoresis processes.

Background

Gel electrophoresis is a powerful tool for separating complex mixtures, identifying molecules, characterizing physical parameters such as molecular weight, and the like. In gel electrophoretic separations, one can use different parameters to enhance the separation between molecules, which may differ in a variety of ways, and to maximize the information one may get from the electrophoresis. Thus, one may use different pore sizes, different temperatures, different buffer systems, different ionic strengths, and the like. In some instances, one can establish gels with gradients, where the gel comprises a range of the particular characteristic. By having gradients, where the response of the sample components are responsive to the gradient and, furthermore, relate to the differences in the gradient in a defined relationship, one can obtain substantially more information from a single gel electrophoresis. There is, therefore, substantial interest in developing techniques which will allow for ease of operation of the gel electrophoresis, while providing for varying conditions for the separation. Relevant Literature

1. Richard P. Haugland, Karen D. Larison (eds.) , Handbook of Fluorescent Probes and Research Chemicals, 5th ed. , Molecular Probes, Eugene, OR p. 129-158 (1992) . 2. A. Chrambach: The Practice of Quantitative Gel Electrophoresis , Advanced Methods in the Biological Sciences (V. Neuhoff, A. Maelicke, eds.), first edition. VCH Publisher, ISBN 0-89573-064-2, Appendix 1, p. 213-222 (1985) . 3. Pier G. Righetti: Isoelectric Focusing: Theory, Methodology and Applications, Elsevier Biomedical Press, Amsterdam, p. 1-386 (1983) .

SUMMARY OF THE INVENTION In accordance with the subject invention, a fluorescent pH-marker is employed in conjunction with a moving-boundary buffer system. The pH gradient may be parallel to or transverse to the line of migration of the sample components. The fluorescent marker allows for detection of buffer boundaries as well as local pH changes.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS In accordance with the subject invention, methods for performing gel electrophoresis employing a fluorescent pH marker are provided, as well as kits, whereby a moving pH boundary may be detected, as well as localized pH. The method involves using at least two different buffering systems, whereby migration of the ions of the buffering system result in changes in the pH within the gel. Thus, one can avoid using a tracking dye, provide for complex separations associated with differences in pH and monitor the variations in pH across the gel in real time.

Various gel compositions may be employed to produce the gel matrix. Polyacrylamide, agarose, gelatin or other gelling or thickening agent may be used for thickening of the gel medium. In standard PAGE technology, gels commonly range between 5-22.5% T (T = total amount of acrylamide or other gelling agent), mostly between 7.5-15% T. Lower percentages may be employed with linear polyacrylamide. In agarose gel electrophoresis, concentrations between about 0.2-2% T may be employed. Also, one may provide for a region which is referred to as the stacking gel, having relatively low polyacrylamide concentration and a resolving gel having a substantially higher acrylamide concentration. Descriptions of forming gels for gel electrophoresis may be found in The Practice of Quantitative Gel Electrophoresis , Chrambach, Vch. Publishers.

The gels will be of conventional thickness and may be of uniform pore size or of a gradient pore size, where the gradient may be in the direction of or transverse to the direction of migration of the sample components.

A wide variety of fluorescent pH markers may be employed. The pH marker may be mobile or be immobilized in the gel, depending upon the nature of the pH marker, the ease with which it may be incorporated into the gel, its effect on forming the gel, and the like. Thus, the pH marker may be conjugated to monomer molecules, which will then become incorporated into the gel, the pH marker may be added to the monomer composition, where it may become immobilized in the gel, or may be included in the buffer, usually the catholyte, so that it migrates through the gel, where it may maintain a substantially uniform concentration. The pH markers of interest should provide for a range of at least about two pH units, preferably at least about three pH units. Desirably, the observed fluorescence will vary over the pH range of interest, so that one can determine the pH at any particular site within about 0.2 pH units. Desirably, the pH marker will be substantially transparent in the visible wavelength range at a pH outside the pH range of interest. For the most part, the pH range of interest will be in the range of about 6 to 10, particularly 7 to 9. The concentration of the pH marker in an electrode buffer will generally range from 100 pg/1 to 10 mg/1, while the concentration in the stacking and separation gel will generally be in the range of about 1 ng/1 to l mg/1.

Illustrative pH markers and their pH detection range include: 7-Hydroxycoumarin (Umbelliferone) , typically pK 7.0-8.5; HPTS (Pyranine) , typically pK 7.2-7.6; Fluoresceins and BCECF, typically pK 5.0-7.0; Rhodols (NERF) , typically pK 4.5-6.0; SNAFL, typically pK 7.0-7.8; SNARF, typically pK 7.0-7.8. As previously indicated, the method will use at least two different buffers, more usually three different buffers. The buffer and the gel in the separation gel may be uniform or may have a gradient, particularly a transverse gradient. Thus, in preparing a gel, one may provide a transverse gradient by either establishing a pH gradient perpendicular to the final separation path using conventional ampholyte distribution obtained by preelectrophoresis of the gel with an electric field applied at a right angle to the direction of the field applied later for separation, or by casting the separation gel using immobilized pH gradients at a right angle to the direction of the field applied later for separation. The buffer in the stacking gel may be different from the separation gel, and/or the catholyte or anolyte buffer. By appropriate choice of the catholyte and anolyte ions, movement of the catholyte and anolyte ions will result in a change in pH as the ions move through the gel. Thus, the ions from the electrode buffer will provide for a moving boundary, which can be observed by virtue of the change in fluorescence of the pH marker. One can also provide for an appropriate anolyte buffer, so that the variation in ions between the catholyte, anolyte and gel buffers provide for a readily-differentiatable pH boundary, as well as a range of pHs across the gel in the direction of migration, and, as appropriate, transverse to the direction of migration. Illustrative systems of buffers and their pH range of interest include: TRIS/Glycine/HCl buffer systems, pH 6.0-9.5; TRIS/Tricine/Acetate buffer systems, pH 4.6-9.2; HEM/ACES/Acetate buffer systems, pH 4.4-8.2; TRIS/Acetate/ETDA buffer systems, pH 7.0-9.0; TRIS/Borate/ EDTA buffer systems, pH 7.5-9.0; Triethanolamine (TEA)/ Glycine/HCl, pH 5.8-9.8; NEM/Taurine/Acetate buffer system, pH 4.6-8.6.

In carrying out a gel electrophoresis, the gel will be prepared as described above, and may or may not comprise the pH marker. The sample would then be applied to the gel at an appropriate site near one end of the gel. The sample may be any one of a variety of combinations of proteins, nucleic acids, charged polysaccharides, or combinations thereof, such as organelles, viruses, etc . , normally including proteins or other variably-charged species. The proteins may have been changed by combination with a detergent, may be reduced, by using a mild reducing agent, or have been subject to other pretreatment. Once the sample has been applied, the sample may then be subjected to an electrical field. In some instances, a stacking gel may be employed which allows for the stacking of the sample in the lane, so that resolution of the sample starts with a sharply-defined band of the sample components. Depending upon the nature of the sample, the conditions of the gel may vary widely as to temperature, electrical field, ionic strength, leading and trailing ion mobility in the buffer composition and the like.

Any convenient means may be employed for monitoring the pH of the gel, continuously or discontinuously. An apparatus which provides for substantially continuous monitoring of fluorescence of a gel is described in U.S. Patent No. 5,104,512, which disclosure is incorporated herein by reference. By continuous monitoring, one can determine the boundary of the migrating buffer, so as to monitor the course of the electrophoresis, as well as monitor local pH changes.

By using pH gradients, one can obtain a pH profile for a protein, so as to provide a specific characteristic of the migratory aptitude of the protein at varying pHs in a single run. In this manner, one can frequently demonstrate separation of proteins, which might otherwise overlap at a single pH, one can provide a characteristic which should vary substantially, even in the case of similar proteins, and one can develop information about the nature of the protein. With the pH marker, one can identify the pH of the sample component in relation to the distance the sample component has migrated. For the convenience of operators, kits may be provided, where the gel may be provided containing a particular pH marker. The kits would provide two or more buffers, to provide a buffer system useful with the particular marker. Other materials which might be included in the kit include reference standards, such as pi markers, and labelling dyes for visualization of sample components, which differ in their spectral response and therefore allow for migration monitoring of sample components without interference with the simultaneous pH monitoring during the experiment.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL pH Tracing to Visualize Stacking and Unstacking in Protein Separations Gel System: 1.5% LE-Agarose

Buffer System: Discontinuous TRIS/HCl/Glycine buffer system containing 0.4% SDS

Buffer Phases:

1. Cathodic Buffer: pH = 8.30

1.44 g Glycine + 0.3 g TRIS/1000 ml 2. Stacking Gel Buffer: pH = 6.90

16 ml 1M H₃PO₄ + 3.57 g TRIS/1000 ml

3. Separation Gel Buffer: pH = 8.90 30 ml 1M HC1 + 22.88 g

TRIS/1000 ml

4. Anodic Buffer: pH = 8.30

1.44 g Glycine + 0.3 g TRIS/1000 ml

(all phases contain 0.4% SDS)

pH-Visualization: Addition of 7-hydroxycoumarin (Umbelliferone) in both, stacking and resolving gel (pK 7.0/8.5) . Dye concentration: 2.5 mg Umbelliferone/250 ml gel (predissolved in 1 ml DMSO)

Operative pH-conditions during electrophoresis:

- pH during stacking: 8.30 - pH during resolving: 9.50

Gel background fluoresces bright blue in alkaline pH range, and is nearly fluorescence-free at acidic stacking conditions showing the buffer front within the stacking gel very distinctively when scanned under UV excitation. Excitation source: UV lamp (280-390 n ) , center wavelength around 305 nm.

Detection: fluorescence emission measurement within visible range (400-705 nm) provides simultaneous monitoring of pH gradient within gel (e.g. blue) and sample component migration {e . g. yellow in this application) .

Sample: Human serum proteins (Protein Control, Sigma) Applied volume: 20 μl

Sample visualization:

Fluorescence labelling with Lucifer Yellow (Molecular Probes) prior to separation: addition of aliquot dye (stock solution 1 μg/μl) + incubation at 37°C for 3-5 min.

Separation conditions: Temperature: 5°C

Sample entry: 5 V/cm; constant voltage; 15 mA max. current Stacking: 15 mA constant current Resolving: 20 V/cm; constant voltage; 50 mA max. current Run time: 65 min. (including stacking) Monitoring buffer front:

Scan interval: 8 min., first scan immediately after sample application. It is evident from the above results, that the subject method and compositions provide for substantial enhancement in the utility of gel electrophoresis. Thus, there is a greatly increased probability, that components of a sample which might otherwise overlap may be separated. A particular pH profile for a component can be defined, so as to provide a distinctive characteristic associated with a particular component. Thus, in many instances, one may be able to identify the particular composition, by virtue of the source of the sample and the pH profile. In addition, one can readily monitor the progress of the electrophoresis by virtue of the pH boundary which can be identified with the change in fluorescence of the pH marker. One can also identify a pH profile associated with a particular sample component.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. In a method for separating variably charged components of a sample by means of gel electrophoresis, whereby a sample is applied to a buffered gel under an electrical field gradient and buffer ions migrate through said gel and said sample components migrate through said gel at a rate proportional to size, charge and conformation, the improvement which comprises: having at least two different buffers among the buffers of the electrodes, separation gel and stacking gel, if present; and a fluorescent pH marker in the buffer in the upstream electrode, in the stacking gel or in the separation gel.

2. A method according to Claim 1, wherein said pH marker is non-diffusibly distributed in said separation gel.

3. A method according to Claim 1, wherein said gel is a polyacrylamide gel.

4. A method according to Claim 1, wherein said separation gel has a transverse pH gradient.

5. A method according to Claim 1, wherein said stacking gel is present and has a different buffer from said separation gel.

6. In a method for separating variably charged components of a sample by means of gel electrophoresis, whereby a sample is applied to a buffered gel under an electrical field gradient and buffer ions migrate through said gel and said sample components migrate through said gel at a rate proportional to size, charge and conformation, the improvement which comprises: having at least two different buffers among the buffers of the electrodes, separation gel and stacking gel, if present; a fluorescent pH marker in the buffer in the upstream electrode, in the stacking gel or in the separation gel; and monitoring the fluorescence of said gel during said separating.

7. A method according to Claim 6, wherein said pH marker is non-diffusibly distributed in said separation gel.

8. A method according to Claim 6, wherein said gel is a polyacrylamide gel.

9. A method according to Claim 6, wherein said separation gel has a transverse pH gradient.

10. A method according to Claim 6, wherein said stacking gel is present and has a different buffer from said separation gel.

11. A gel electrophoresis kit comprising: an electrophoresis gel comprising a buffer, a fluorescent pH marker, an electrode buffer different from said electrophoresis gel buffer or said gel comprises a stacking gel and a separation gel, wherein said stacking gel and said separation have different buffers.

12. A kit according to Claim 11, wherein said fluorescent pH marker is non-diffusibly bound in said separation gel.