US3355372A - System, apparatus, and method for the removal of background stain by means of electric current from an electrophoresis supporting medium - Google Patents

Description

Nov. 28, 1967 SYSTEM, APPARA'riJs BACKGROUND STAIN T G. FERRIS ETAL AND METHOD FOR THE REMOVAL BY MEANS OF ELECTRIC CURRENT FROM AN ELECTROPHORESIS SUPPORTING MEDIUM Filed March 6, 1963 2 Sheets-Sheet l INVENTORS THOMAS G. FERRIS Nov. 28, 1967 T. G. FERRIS ETAL 3,355,372

SYSTEM, APPARATUS, AND METHOD FOR THE REMOVAL OF BACKGROUND STAIN BY MEANS OF ELECTRIC CURRENT FROM AN ELECTROPHORESIS SUPPORTING MEDIUM Filed March 6, 1963 2 Sheets-Sheet 2 INVENTORS THOMAS G. FERRIS ROBERT E. EASTERLING RICHARD E. BUDD by @n A 7' TO/PNEV United States Patent SYSTEM, APPARATUS, AND NETHOD FOR THE REMOVAL OF BACKGROUND STAIN BY MEANS OF ELECTRIC CURRENT FROM AN ELECTRO- PHORESIS SUPPORTING MEDIUM Thomas G. Ferris, 5420 Alta Vista Road, Bethesda, Md. 20014; Richard E. Budd, 659 Wildwood Blvd., Williamsport, Pa. 17701; and Robert E. Easterling, 721 Courtland Drive {P.O. Box 711), Sanford, N.C. 27330 Filed Mar. 6, 1963, Ser. No. 263,361 Claims. (Cl. 204-180) The invention described herein may be manufactured and used by or for the Government of the United States of America for governmental purposes without the payment of any royalties thereon or therefor.

This invention relates to a system, apparatus, and method for the removal of background stain by means of electric current from an electrophoresis supporting medium.

A most common prior art method for removing background stain from an electrophoresis supporting medium used for the fractionation, for example, of serum proteins, has been by washing techniques, often by circulating washing techniques. In certain cases, such as when polymerized acrylamide gel is the medium, the removal of the background stain by washing is quite time-consuming, requiring perhaps from two to five hours to complete. Arrangements for the removal of background stain by means of electric current have been published as indicated, for example, in the article entitled, Electrophoresis of Serum Protein in Acrylamide Gel, by the present inventors, Ferris, Easterling, and Budd, appearing in the American Journal of Clinical Pathology, vol. 38, No. 4, pages 383- 387, October 1962, and ir1,the Instruction Bulletins for Disc Electrophoresis distributed by Canal Industrial Corporation, Bethesda, Md. However, these arrangements are in the one case limited to small pieces of gel, and in both cases are apt to suffer from certain deficiencies.

The deficiencies encountered with horizontal destaining apparatus, such as that described in the aforementioned article of the inventors, are due primarily to the evolution of gas at the electrodes. This gas is sometimes trapped beneath the gel, interfering with the uniform and complete removal of the background stain. When the gel is in direct contact with the anode during destaining, bubbles form within the gel. These bubbles interfere with subsequent scanning of the gel in a recording densitometer. In horizontal destaining apparatus when the gel to be destained is smaller than the electrode plates, c.g., when the gel has been sliced into strips containing individual electrophoretic patterns prior to destaining, the current tends to shunt around the gel rather than passing through it. As a result, the destaining of the gel is inefiicient and often incomplete, even though destaining is carried out beyond the thirty minutes which is routine with the present invention.

An object of the present invention, therefore, vide a system, apparatus, and method for the electrical destaining of an electrophoresis supporting medium which rapidly and efiectively removes the background stain and leaves clearly defined electrophoretic patterns with an adequately clear background in the electrophoresis medium to facilitate accurate scanning and evaluation of the electrophoretic patterns.

Other objects, advantages and novel features of the invention will become apparent from the following detailed description when considered in conjunction with the accompanying drawings wherein:

FIG. 1 is an exploded elevation view of a preferred embodiment of the apparatus of the invention;

is to pro- FIG. 2 is an axonometric, generally top view of a slab of gel in generally horizontal position adhered to a piece of filter paper;

FIG. 3 is a vertical cross section taken along the line 3-3 in FIG. 4 of the apparatus of FIG. 1 assembled and with the gel-filter paper laminate of FIG. 2 in position for destaining; and

FIG. 4 is a cross section taken along the line 4-4 in FIG. 3.

Reference is now made to the drawings. The numeral 2 indicates a container which for convenience can be an ordinary cylindrical battery jar. Lid 4, conveniently of clear plastic, is provided to insure good visibility of the contents of the container and to protect against the danger of accidental electric shock. The lid is provided with holes 6 through which can be inserted insulated connectors 8 and 10 for connecting to a source of electricity the prongs 12 and 14 of the anode 16 and cathode 18, respectively. The cylindrical electrodes 16, 18 are held in fixed, generally concentric relation to each other by any convenient means, such as a press fit, serving to fasten them to an insulating base 20. Typically, the anode 16 may be tightly sweated onto the base 20, readily made of plastic, with such a good fit that liquid such as the electrolyte 22 is contained in the annular space between the two electrodes, the container 2 then serving merely as a precaution against leakage.

In FIG. 2 there is shown a slab 24 of electrophoresis supporting medium, such as polymerized acrylamide gel, adhered to a sheet of filter paper 26 to form a laminate. In FIGS. 1, 3 and 4 the lam nate is shown as having been rolled into a cylinder with the slab of gel on its interior and the filter paper being fastened at its overlapping ends by such convenient means as staples 28. Bafile 30 of insulating material is shown transversely spanning those lines of the electric field between the two electrodes which do not intersect the electrophoresis supporting medium.

To facilitate an understanding of the system apparatus and method of the invention, a description of the invention is given herewith as applied, by way of example only, to the destaining of a particular gel used in serum protein electrophoresis. To provide an informative background setting to elucidate the use of the invention, a brief description is given of the preparation of the electrophoresis supporting medium.

Serum protein electrophoresis is carried out in a vertical electrophoresis cell in the manner mentioned in the previously referenced article in the American Journal of Clinical Pathology using a polymerized acrylamide gel in the manner analogous to that used for hemoglobin electrophoresis described in the article by Ferris, Easterling, and Budd, appearing in Blood Volume 19, No. 4, April 1962, pages 479-481. Although other supporting media for zone electrophoresis can be used, the one chosen for this example is the acrylamide gel known as Cyanogum 41 described in the article by Raymond and Weintraub published in Science, Sept. 18, 1959, page 711, and in the bulletin published by the American Cyanamid Company entitled, Cyanogum 41 Gel-ling Agent for Industrial Use. After electrophoresis is completed, the gel is carefully removed from the cell and immediately placed in a staining solution. The gel may typically be 5" x 7" x mm. thick. Various stains can be used, but an excellent stain for serum proteins is the stain known as Amidoschwartz 10B, also known as Buffalo Black NBR, Naphthol Blue Black and Acid Black I, appearing in the Color Index under CI20470. A stock stain solution can be made of one gram of Amidoschwartz 10B dissolved in ml. methanol to which is added 700 ml. glacial acetic acid and qs to one liter with distilled water. A working stain solution is made by diluting the stock solution 1:1 with distilled water just prior to use.

T he protein is fixed in the gel and stained simultane- "ous by this procedure. It is necessary to leave the gel in the stain long enough to completely fix and stain the lproteins. If the gel is removed prematurely, part of the Eprotein will not be fixed and will be removed from the :eel along the background stain, during the destaining lproce'ss. For convenience, the gel can be left in the work- :in'g stain overnight (16 hours). Staining and fixing can e carried out in 2 hours if the gel is placed in the stock stain solution instead of the working stain solution.

After the staining-fixing is completed, the gel is carefully removed from the stain solution and laid out on a fiat, smooth surface, such as a piece of glass. A piece of heavy dry filter paper, preferably somewhat larger than the gel is gently pressed onto the gel as shown in FIG. 2. The gel and filter paper will adhere and will remain adhered even when the paper is held in a vertical position. The filter paper used is a heavy filter paper which remains rigid when wet. The filter paper-gel laminate is formed into a cylinder with the gel on the inside of the paper cylinder. The laminate is retained in the cylinder form by any convenient means such as by overlapping the ends of the filter paper and stapling the filter paper together in several places along the seam as shown in FIGS. 1, 3 and 4. The cylinder laminate is then carefully placed in the destaining apparatus and takes the position shown in F165. 3 and 4. To proceed with the setup of the system of the invention, the apparatus is filled with sufiicient 115% acetic acid solution to bring the electrolyte level just over the upper edge of the gel. The'concentration is not critical, but best results are obtained with acetic acid solutions in therange of 1G%l5%. Other electrolytes can be used, but acetic acid is a logical choice since acetic acid is used to fix the protein during the staining-fixing part of the procedure and its molecule is small permitting rapid penetration of the gel.

The paper-gel laminate cylinder is adjusted so that it is approximately concentric with the apparatus and the insulating baffle shown in FIGS. 1, 3 and 4 is placed to prevent the shunting of current through the area between the ends of the gel, permitting more efficient destaining of the gel. The insulating bafile is not absolutely essential for destaining, but its use appreciably improves efiiciency. If the ends of the gel areapproximated, so that the cylinder of gel is'essentially continuous and without a gap such as seen in FIG. 4, then the bafiie is unnecessary. Such a configuration can be achieved by, for example, placing the filter paper with one ultimately vertical edge flush with an ultimately vertical edge of the gel instead of the two opposite edges of the paper overhanging the gel, as in the illustrated embodiment. The laminate cylinder would then be formed by rolling the laminate into a position with the gel ends abutting and with the single projecting end of filter paper overlapping,

on the outside of the cylinder, its opposite end which is flush with the gel surface. The overlapping portions of filter paper would then be fastened together by any suitable fastening mean s, such as adhesive or tape, which remain fastened when wet outdo not hinder the electrical .destaining process.

The lid 4 is now placed on the container 2, and the leads 3 and 10 from the direct current power supply :are attached to the prongs 12 and 14 so that the outer electrode 16 is the anode, Various combinations of time and current can be used for destaining. With a gel slab of the aforementioned size, a constant current of one :ampere applied for thirty minutes has been found to produce excellent results. The current applied is reduced in proportion to the reduction in gel area. For example, if a piece of gel to be destained is half the Width of the aforementioned uncut gel, then the current applied would .be 0.5 ampere instead of 1 ampere used to destain the 4 whole gel slab. *If a slab of gel, alonewithout filter aper backing, is formed into a cylinder the curvature of the gel gives it sufficient rigidity to stand upright in the electrolyte. However, under such circumstances, the gel tends to cling to theouterelectrode and although it can be satisfactorily destained by this procedure, bubbles form within the gel when the gel is in direct contact with the anode during the destaining. These bubbles interfere, as previously noted, with subsequent scanning of the gel in a recording densitometer. In addition, the gel tends to be slippery and hard'to handle, and it is sometimes difficult to get the gel into the apparatus without tearing it. The filter paper cylinder technique solves both the problem of gel support and gel positioning between the two electrodes. The paper cylinder also provides a convenient means of getting the gel into and out of the apparatus without damage to the gel. It has the additional advantage of acting as a barrier to the circulation of removed stain throughout the electrolyte. That is, with the'electric current passing'between the two electrodes, the stain is pulled out of the gel and toward the anode 16 by the electric current; and as the stain passes through the paper cylinder and into the electrolyte, it is trappedin the annular space between the wall of the anode and the paper..

As the destaining progresses, the electrolyte between the paper and the anode becomes very dark with removed stain while the electrolytebetweenthe paper-gel laminate and the cathode remains relatively clear. The light coming through the transparent bottom of the apparatus makes the gel readily visible. As destaining proceeds, the gel can be seen to become transparent as the background stain is removed by the electric current.

After destaining is completed, the current is shut off and the leads 8 and 10 disconnected. The paper-gel laminate cylinder is carefully removed from the apparatus and placed in a shallow pan of water. The paper is cut apart along the stapled seam and the laminate is. permitted to lie flat in the water, gel side down. The filter paper can now be easily removed by gently pulling the paper away from the gel while the gel remains submerged in the water. I v

Although the invention has been illustrated as using acrylamide gel, it is also applicable to other electrophoresis "supporting media such, for example, as starchgel.

Obviously, many modifications and variations of the present invention are possible in the light of'the above teachings. It is therefore to be understood.'that within the scope of the appended claims the invention may be practiced otherwise than as specifically described.

What is claimed is: f

1. A method of removing stain from an electrophoresis supporting medium comprised of 'a slab of gel which includes: l

adhering said slab of gel to filter'paper,

configuring the laminate formed of said gel and, filter paper into a generally cylindrical shape with the gel on the interior thereof, H v fastening the adjacent end portions of the filter paper together to retain the. laminate in its generally cylindrical,relatively rigid form, disposing said laminate generally vertically concen-v trically in electrolyte between a pair of generally concentrically spaced generally cylindrical electrodes and free of contact with each electrode, maintaining the electrolyte at a level essentially just covering the upper edge of said gel, and

applying a direct electric potential difference between said electrodes to attract stain from said gel through said filter paper toward the outer electrode.

2. The method of claim 1 wherein said gel comprises polymerized acrylamide. v

3. The method of claim 1 further including the step of interposing during the application of electric potential difference, bafiic means between said electrodes spanning transversely those radii of the radial electric path through the electrolyte between said electrodes which do not intersect said electrophoresis, supporting medium.

4. A method of removing stain from an electrophoresis supporting medium comprised of a slab of gel which includes:

adhering said slab of laminate;

disposing said laminate generally vertically in electrolyte between, but free of contact with, a pair of electrodes, the surfaces of the electrodes being generally parallel to the gel-paper interface of said laminate; and

applying a direct electric potential ditterence between said electrodes to attract stain from said gel toward one of said electrodes.

5. The method of claim 4 wherein the gel is polymerized acrylamide.

gel to filter paper to form a References Cited UNITED STATES PATENTS 1,235,064 7/ 1917 Schwerin 204-480 1,266,329 6/1918 Schwerin 204180 6 1,815,302 7/1931 Hogstad 204-480 1,878,235 9/1932 Gortner et a1. 204180 2,741,595 4/1956 Juda 204301 OTHER REFERENCES Barka: The Journal of Histochemistry and Cytochernistry, Studies of Acid Phosphatase, I. Electrophoretic Separation of Acid Phosphatase of Rat Liver on Poiyacrylamide Gels, vol. 9, No. 5, September 1961, pp. 542547.

Smithies: J. Biochemistry, An Improved Procedure for Starch Gel Electrophoreses; Further Variations in the Serum Proteins of Normal Individuals, vol. 71, 1959, pp. 585587.

Heftman: Chromatography, pp. 262-265, 1961.

Raymond: Analytical Biochemistry, Preparation and Properties of Acrylarnide Gel for use in Electrophoresis, pp. 391-396, vol. 1, 1960.

JOHN H. MACK, Primary Examiner. HOWARD S. WILLIAMS, Examiner. J. BATTIST, E. ZAGARELLA, Assistant Examiners.

Claims (1)

1. A METHOD OF REMOVING STAIN FROM AN ELECTROPHORESIS SUPPORTING MEDIUM COMPRISED OF A SLAB OF GEL WHICH INCLUDES: ADHERING SAID SLAB OF GEL TO FILTER PAPER, CONFIGURING THE LAMINATE FORMED OF SAID GEL AND FILTER PAPER INTO A GENERALLY CYLINDRICAL SHPAE WITH THE GEL ON THE INTERIOR THEREOF, FASTENING THE ADJACENT END PORTIONS OF THE FILTER PAPER TOGETHER TO RETAIN THE LAMINATE GENERLLY VERTICALLY CONCENTRICALLY IN ELECTROLYTE BETWEEN A PAIR OF GENERALLY CONCENTRICALLY SPACED GENERALLY CYLINDRICAL ELETRODES AND FREE OF CONTACT WITH EACH ELECTRODE, MAINTAINING THE LEECTROLYTE AT A LEVEL ESSENTIALLY JUST COVERING THE UPPER EDGE OF SAID GEL, AND APPLYING A DIRECT ELETRIC POTENTIAL DIFFERENCE BETWEEN SAID ELECTRODES DO ATTRACT STAIN FROM SAID GEL THROUGH SAID FILTER PAPER TOWARD THE OUTER ELECTRODE.

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