US3497441A

US3497441A - Electrophoresis system and method

Info

Publication number: US3497441A
Application number: US638815A
Authority: US
Inventors: Joseph Paksi
Original assignee: Individual
Current assignee: Individual
Priority date: 1967-05-16
Filing date: 1967-05-16
Publication date: 1970-02-24
Anticipated expiration: 1987-02-24

Description

Feb.24,1 970 J in. 3,497,441

ELECTROPHORESIS SYSTEM AND METHOD Filed May 16, 1967 2 sheets-sheet 1 Fig. 4

INVENTOR.

JOSE/9H PAKS/ BY WM, Q/Og/MMW 3W M460 ATT QMEYs.

J. PAKSI ELEOTROPHORESIS SYSTEM AND METHOD Feb. 24, 1970 2 Sheets-Sheet 2 Filed May 16, 1967 INVENTOR. v JOSE/ H PAKSI BY v a ATTORNEYS.

m a /wq United States Patent 7" 3,497,441 ELECTROPHORESIS SYSTEM AND METHOD Joseph Palrsi, 2217 Westminster Road, Cleveland Heights, Ohio 44118 Filed May 16, 1967, Ser. No. 638,815 Int. 'Cl. B01k /00 US. Cl. 20418fl 9 Claims ABSTRACT OF THE DISCLOSURE An electrophoresis system for separation analysis of solutions of ionized particles. A starch-gel medium is formed on a Mylar substrate and separated by channels into test strips. Under an electric current, particles of a sample material migrate different distances through the medium. The strips can be scanned in photoelectric analyzers. A drum applicator and cutting board facilitate preparation.

BACKGROUND OF THE INVENTION Field of the invention This invention relates to electrophoresis, which is the migration of particles under the influence of an electric field. More particularly, this invention relates to methods and apparatus for utilizing electrophoresis to analyze solutions of ionized particles, especially biochemical materials.

Description of the prior art Electrophoresis is a known and important analytical technique. It has found particular usefulness in the medical field because it provides a means of performing the separation of proteins, amino acids, nucleic acids, peptides, and other important biochemical materials. Four known types of electrophoresis are: (1) moving boundary electrophoresis, (2) zone electrophoresis, (3) two dimensional electrophoresis, and (4) immunoelectrophoresis.

Moving boundary electrophoresis permits quantitative estimation of the mobilities and concentrations of various components, as in a mixed protein solution, such as plasma. Physical channels are provided for the solution and .movement of the particles in the solution is followed when an electric current is past through the material.

Zone electrophoresis uses a supporting medium in which ions of the solution migrate and separate into zones, depending upon their mobility in an electric field. As compared with moving boundary techniques, the amount of material to be analyzed is very small. Turbid or highly pigmented samples cannot be satisfactorily illuminated for moving boundary electrophoresis. No such problem arises with zone electrophoresis techniques.

Two dimension electrophoresis combines two methods of zone electrophoresis into a single method to provide a more complete resolution of the components of the material being tested. Basically, components are separated in a first direction, then transferred to a second medium, where the electrophoresis is continued at a right angle to the previous one for additional time.

Immunoelectrophoresis is used to study the complexity of antigens or antibodies through the use of zone electrophoretic techniques.

The present invention relates to zone electrophoresis, and many types of zone electrophoresis are already known. These known types of zone electrophoresis include 1) paper electrophoresis, (2) cellulose acetate 3,497,441 Patented Feb. 24, 1970 membrane electrophoresis, (3) gel electrophoresis, including agar-gel, polyacrylamide gel and starch-gel block electrophoresis, and (4) preparative electrophoresis, including starch block, sponge rubber, and continuous flow electrophoresis. The different types of zone electrophoresis techniques indicate the media in which the material to be analyzed migrates. These different techniques vary in their sensitivity, convenience, and speed. For example, in normal human serum, five protein fractions can be identified with paper electrophoresis and nine with cellulose acetate membrane electrophoresis, the essential differences in the techniques and the apparatus employed being the result of the type and texture of the supporting medium in those two instances.

Agar-gel electrophoresis provides a technique that is simple, rapid, has high resolving power and demonstrates the relative mobilities of the migrating proteins. With this technique, tissue fragments can be studied electrophoretically, and the agar-gel provides a sensitive technique for immunoelectrophoresis. Five protein fractions can be identified from normal human serum with this technique.

In disc electrophoresis, advance preparation of a polyacrylamide gel in three stages is necessary. The three layers, the top one of which includes the specimen, are placed in a tube and photopolymerized. Twenty protein fractions of human serum can be identified with this method.

In starch-gel block electrophoresis, the migrating ions must pass through the gel matrix, rather than in aqueous film around the supporting material, as in the filter paper system. Starch-gel hinders the migration of larger proteins more than smaller proteins. Exact and precise preparation of the gel is essential and the nature of the starch itself is of primary importance. After electrophoresis, the

" gel is cut into thin layers and stained, then observed or scanned. In normal human serum, 20 protein fractions can be identified with this method.

Starch block electrophoresis uses a homogeneous block of potato starch. The material to be fractionated is applied in a narrow slit and the block is covered with a paraffin film to prevent evaporation. After electrophoresis, the block is cut into short segments, the material eluted, and analyzed. This technique is most useful for recovering fractions of large molecular weight, which migrate freely in starch block, and which are unable to pass through the mesh of gels. In normal human serum, five protein fractions can be identified with this method.

Sponge rubber electrophoresis provides a simple method of separating protein components of serum. The supporting medium does not complex with serum proteins and the sponge can be squeezed to obtain the proa. teins after separating. -Five protein fractions can be identified from normal human serum with this method.

In continuous flow electrophoresis, electric current is continuously applied and the sample is continuously applied to the medium used, normally filter paper. The electromotive force is applied transverse to the sample track, and as the sample travels in the medium, it spreads fanwise. The study normally takes several days, and five protein fractions of normal human serum can be identified.

SUMMARY OF THE INVENTION The present invention is directed to zone electrophoresis using a medium coating on a plastic film base. It specifically utilizes a thin layer of starch-gel medium adhered to an optically transparent Mylar film base. This film base has been selected from over 100 materials ranging from paper through glass and various types of plastic, and has been found to have a superior ability to adhere the starch-gel medium in a desirably thin coating. This arrangement provides a test strip of starch-gel medium, which has greater sensitivity than paper or cellulose acetate media, while at the same time afiording the convenience of automatic scanning of the test strip for quantitative analysis of the sample material being tested.

As indicated above, know types of starch-gel media have been used in block form, which is quite thick. Approximately 8 hours are required between the making of the gel and its use for testing. With the present system, however, the starch-gel on Mylar strip can be used within minutes from the time preparation is begun. Moreover, the known starch-gel blocks could not be scanned, whereas the present strips lend themselves to the use of known photoelectric scanning techniques.

The Mylar base and starch-gel medium of this invention provide a matrix in which a density pattern of sample particles is developed from which the relative percentages of different fractions in a sample material, such as proteins that make up a serum, can be calculated. Moreover, with this starch'gel and Mylar base medium, to 25 fractions can be separated from normal serum, as compared with 5 fractions possible with a presently used paper media, of comparable convenience. Also, a plurality of samples can be handled on a single sheet or film base.

In addition to the above, the present invention provides specific apparatus and techniques for facilitating the preparation, use, and scanning of a film supported medium. To this end, a drum or roller-type applicator is provided to assist in the coating of the Mylar base with a thin film of starch-gel. A cutter board of novel construction is provided for preparing the coated base and medium into usable strips for electrophoresis. Also, a technique for glycerinating the starch-gel has been developed to inhibit the tearing of the starch-gel film, maintain the gel in adherence to the base film, and harden the gel so that the finished strip can be run through an automatic scanner for analysis. A mat strip has been provided for use with the starch-gel film to accommodate the conventional photoelectric scanners, which require diffused light.

Basically, in the preferred mode of practicing this invention, a sheet of Mylar is coated with a thin film of starch-gel. The coated film is placed on a cutting board and parallel channels are for-med to divide the gel coating into strips. The gel coated film is then placed on a support stand of an electrophoresis cell with one end of the film connected to a negative potential and another portion of the film spaced therefrom connected to a positive potential. A sample is applied to each strip of the gel at a starting line, and a current is applied across the film. After a predetermined time during which the particles of the sample, such as serum proteins, migrate, the film is removed. The sheet is then stained, e.g., with protein stain. It is then destained to take out the background color leaving the dye bound only to the different fractions of the sample material in the medium. To facilitate handling, especially in automatic scanning equipment, the starch gel is glycerinated in accordance with a critical technique to be described in detail subsequently. The film is then cut into strips along the channels previously made in the gel, to facilitate scanning in an automatic photocell analyzer. A matte strip is placed over the gel to diffuse lightpassing through the optically clear Mylar film base, and the sandwich is fed through the analyzer. The dyed protein bands in the gel vary the amount of light passing through the strip. The light transmitted indicates the quantity and location of the various parts of the sample, which have migrated different distances during the time that the strip was in the electrophoresis cell.

Accordingly, it is an object of this invention to provide new and improved methods and apparatus of electrophoresis that are convenient to use and sensitive in operation. Other objects and a more complete understanding of this invention will be obtained from the detailed description, when considered in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a top plan view of an applicator for applying starch-gel to a sheet of plastic film;

FIGURE 2 is a sectional view of the applicator of FIGURE 1, taken along the line 22 and looking in the direction of the arrows;

FIGURE 3 is a top plan view of a cutting board for preparing a coated sheet of plastic film for use with the present system;

FIGURE 4 is a longitudinal sectional view of the cutting board of FIGURE 3, taken along the line 44 and in looking in the direction of the arrows;

FIGURE 5 is a diagrammatic view of a pipette and vacuum source, used in the preparation of a coated plastic film for use with the present system;

FIGURE 6 is a diagrammatic perspective view of a sheet of plastic film to be coated with a layer of starch-gel for use in the present system;

FIGURE 7 is a narrow plastic strip with a matte or diffusing surface for use in the present system;

FIGURE 8 is a side elevational view, with parts removed and parts in section, diagrammatically illustrating a plastic sheet wrapped around the applicator of FIG- URE 1;

FIGURE 9 is a diagrammatic view similar to FIG- URE 8, showing the manner in which a plastic sheet is removed from the applicator of FIGURE 8 to coat the sheet with a layer of starch-gel;

FIGURE 10 is a diagrammatic perspective view of the cutting board of FIGURE 3 and pipette of FIGURE 5, illustrating the manner in which the coating on a sheet of plastic film is divided into spaced strips in preparation for use;

FIGURE 11 is a diagrammatic perspective view of a coated sheet of plastic film prepared in the manner illustrated in FIGURE 10;

FIGURE 12 is a transverse sectional view of the coated sheet of FIGURE 11, taken along the line 12-12 and looking in the direction of the arrows;

FIGURE 13 is a diagrammatic perspective view of an electrophroesis cell for applying an electric current to the starch-gel film of FIG-URE 11; and

FIG-URE 14 is a diagrammatic view, partly in Section, illustrating the manner in which a strip of the film in FIGURE 11 along with the matte strip of FIGURE 7, are automatically scanned to determine density variations of dyed materials that have migrated within the layer of starch-gel adhered to the plastic film.

DESCRIPTION OF THE PREFERRED EMBODIMENT An applicator 20 is shown in FIGURE 1 for applying a coating of gel medium to a base sheet of plastic film. The applicator 20 consists of a drum 22 about which a plastic sheet will be wrapped for coating, a vessel 24 that holds a medium to be applied to the plastic sheet and which also receives a portion of the drum 22, and two

end plates

26, 27 that support the drum and vessel. Preferably, the vessel is made of plastic that will withstand temperatures of 200 to 210 degrees Fahrenheit at which starch-gel medium may be when placed in the vessel. As best indicated in FIGURE 2, the vessel 24 is formed of a longitudinal segment of a cylindrical tube, oriented concave upward and extending between the two

end plates

26, 27. The vessel is adhered to the two end plates and together with the end plates forms a support for the drum 22.

The drum 22 is cylindrical in shape, being in the form of a tube with circular end walls 29, 30. A stud shaft 31 extends from the end wall 29, and a stud shaft 32 extends from the end Wall 30. These stud shafts are received in semi-circular recesses at the top of

end plates

26, 27, one such recess being located at 34 in FIGURE 2. The drum 22 is supported by the

end plates

26, 27 so that the spacing between the drum 22 and vessel 24 is somewhat greater at a central area 24c of the vessel than at

longitudinal edges

24a, 24b. In use, the drum is rotated in a direction from the vessel edge 24a to edge 24b and the greater spacing in the central area acts as a reservoir for the coating medium. The close spacing at 24b prevents spillage and helps control the coating thickness.

A cutting board 38 is shown in FIGURE 3 for use in dividing the medium coated on a sheet of plastic film into spaced strips and later for cutting the film into strips. The cutting board 38 is preferably of plastic and consists of a fiat rectangular panel 40 adapted to receive a coated sheet of plastic film in a flat condition. A raised border 42 is provided about three sides of the fiat rectangular panel 40, and includes three portions 42a, b, c. The panel has one open end at 40a. The surface of the panel 40, within the border 42, is constructed of a size to receive a sheet of plastic film material coated on the applicator 20. A removable cross piece 44 extends across the panel 40 and rests on the

border portion

42a, 42c, and is spaced from the border portion 42b and the open end 40a. Locating studs 45 on the border portions 42a, 0, are received in apertures in the cross piece and locate it relative to the top border portion 42b. As shown in FIG- URE 3, the cross piece 44 and the border portion 42b are adapted to support a T-square 46. The T-square 46 will slide across the board 38 above the panel 40, and is used to prepare lines in the coating of the plastic film that is placed on the panel 40.

A clamping member 48 is located at the open end 40a of the panel 40, and is used for cutting the coated film into separate strips after electrophoresis. The clamping member 48 includes a flat plate 49 that extends across the panel surface 40, and fits closely within the border portions 4211, 420. The fiat plate 49 is relatively narrow and is thinner than the thickness of the raised borders 32a, 42c so that it can be supported flush with the raised borders and yet provide space between it and the upper surface of the panel 40 to receive a sheet of plastic film material coated with a medium, such as a starch-gel. A rod 50 of circular cross section extends longitudinally d Wn the middle of the fiat plate 49 and across the panel 40. Opposite ends of the rod 50 rest upon raised

border portions

42a, 42a. The rod 50 is secured to the upper surface of the flat plate 49 and thereby supports the plate above the panel 40. Spaced pairs of

blocks

51, 52 on the border portions 42a, 420, respectively, locate the extending ends of the rod 50 to maintain the clamping member 48 in proper location, but pivotable about the axis of the supporting rod 50. When the clamping member 48 is pivoted about the axis of the rod 50, it presses against the film on the panel 40, to securely hold the film during a cutting operation. It will then pivot to a horizontal position, to allow the film to be moved relative to the panel 40.

A channel former 56 is shown in FIGURE 5, and is used to make channels in the medium coated on the sheet of plastic film used in the present system. The channel former 56 is preferably a glass capillary pipette 58 connected by a vacuum hose 59 to a vacuum source, such as a vacuum pump 60. The pipette 58 has a small open end 6.1 approximately 1 millimeter in diameter that can be guided along the T-square 46 of the cutting board 38 to form channels in a medium on a plastic film, as will be explained in more detail subsequently in connection with the method and operation of the present system.

A sheet of film 66 is shown in FIGURE 6 of the drawings, and is constructed to be coated with a medium in which particles can migrate under the influence of an electrical field. When coated, it is used to separate particles by electrophoresis.

The film 66 is formed of two separate pieces, a large piece 67 to be coated and small manipulator piece 68. The large and small pieces are of equal width, and abut each other as illustrated. A strip of tape, for example, cellophane adhesive tape 69 extends across the upper surface in the orientation of FIGURE 6, and adheres the two

pieces

67, 68 together. The large piece 67 when coated is used to provide several narrow strips for re ceiving samples of materials to be analyzed. The smaller piece 68 is used as a leader to facilitate coating the larger piece on the applicator 20 of FIGURES 1 and 2. The width of the supporting film 66 corresponds essentially to the width of the panel 40 within the raised border portions 42. A width suitable for making a plurality of smaller strips is preferred, for example a width of 11 /2 inches is particularly suitable for providing five spaced test strips and can be received on existing equipment for applying a current across the gel. Suitably, the length of the large piece 67 can be 14 /2 inches and the length of the second smaller piece can be 5 inches.

For optimum results in separating ions of diiferent mobility, in accordance with this invention, a starch-gel medium is used. With this preferred medium, only a supporting film 67 made of polyethylene terephthalate, such as Mylar, has been found to be completely suitable. The discovery of the suitability of this film material, its use to support a starch-gel medium, and its superiority over other possible substrates for supporting a layer of starch-gel for electrophoresis constitutes an important aspect of this invention. This Mylar film material is optically clear, strong, and tenaciously adheres a medium of starch-gel especially suitable for the present pr cess. Preferably, a sheet of film 0.005 inch thick is used.

A narrow plastic strip 70 is shown in FIGURE 7 for use with test strips prepared from the large piece 67 of the supporting film 66 when the large piece is cut into separate test strips and analyzed after electrophoresis. The strip 70 preferably has one surface 71 of a matte finish, or the strip is otherwise translucent. When this strip is placed over the medium on the optically clear supporting film 67, it diffuses light and facilitates scanning by photoelectric detectors that are constructed to operate with diffused light. Preferably, the strip 70 is of polyethylene terephthalate, such as Mylar.

The manner in which the above-described apparatus is used and the method of practicing the present invention is best understood in connection with FIGURES 8 to 14 of the drawings.

The drum 22 is removed from the applicator 20 and the composite supporting film 66 is fastened to the drum, as by a piece of cellophane adhesive tape. The supporting film is attached at one end of the large piece 67 and is then rolled about the drum, so that the small manipulator piece 68 forms a partial cover over the large piece 67 on the drum. The outer end of the manipulator piece 68 is then adhered to the rolled film, as by a small piece of adhesive tape to hold the film wrapped about the drum.

A starch-gel medium is prepared and placed in the vessel 24. The medium is shown in the vessel 24 in FIG- URE 8, and indicated by the reference numeral 75. The drum 22 with the rolled supporting film 66 is then placed within the vessel 24, supported on the two

end plates

26, 27 so as to be spaced from the vessel 24 but partially immersed in the starch-gel 75. When the drum 22 is placed into position, the terminal end of the film 66 (i.e., the outer end of the manipulator piece 68) is located out of the gel, and pointing upward, as illustrated in FIG- URE 8. The outer end is then loosened from the drum 22, and pulled around and over the drum, as illustrated in FIGURE 9. As a result of this manipulation, the surface of the film 66 facing the drum 22 remains free of starch-gel, while the outer surface is coated with a continuous layer 76 of starch-gel of essentially uniform thickness preferably 1 millimeter thick. The manipulator piece 68 serves to initially remove any air bubbles in the starch-gel so the coating on the piece 67 is uniform.

By way of a specific example, the starch-gel 75 with which the sheet or film 66 is coated is prepared by diluting 15 ml. concentrated Tris Buffer (No. 301), manufactured by The Spel Systems, Cleveland, Ohio, with 135 ml. of distilled water, and mixing the diluted butter with 18 grams Connaught Hydrolyzed Starch Powder in a two liter round bottom fiask so that the starch is evenly distributed. The starch is heated to the boiling point with constant swirling. The boiling is continued for a few seconds only, and the flask is then connected to a water pump to remove all air bubbles. This entire procedure takes less than 2 minutes, consisting of 70 seconds heating plus 20 seconds degassing. The hot gel at about 207 degrees Fahrenheit is then poured into the vessel 24 at an even rate to avoid entrapping air bubbles. The drum 22 wrapped with the film 66 is then placed into position in the vessel so that the outer end of the film is facing up ward about /2 inch from the gel surface. The tape adher ing the outer end of the film to the drum is removed and the film is rolled off the drum with constant speed and pulled onto a horizontal, smooth, surface. The upper surface only of the film is coated with the gel to a thickness of 1 millimeter. After one minute, the tape 69 connecting the small piece 68 to the large piece 67 is removed and the small piece of film is discarded. The gel on the film is now ready to be divided into spaced strips.

To prepare the layer of gel 76 on the film 67, portions of the gel are removed to form channels 78 in a pattern that is best shown in FIGURE 11 where spaced test strips 79a, b, c, d, e are shown separated by narrower strips 80, all defined by the channel '78. As shown, the channels 78 do not extend the full length of the film 67. In practice the length of the channel 78 depends upon the length of strip 79 desired, which in turn Will depend upon the size and construction of the electrophoresis cell or apparatus used to apply a current to the gel medium on the film.

The manner in which the channels 78 are formed is shown in FIGURE of the drawings. The supporting film 67 with the layer of gel 76 is placed on the panel 40 of the cutting board 38. The film is located within the raised border 42 on three sides of the cutting board, and extends slightly from the open end 40a. The cross piece 44 is then placed across the border portions 42a, 420. The T- square 46 is positioned to extend along the cutting board 40, resting on the raised border 42b and the cross piece 44. If desired, the flat rectangular panel 40 can be lined or a pattern can be placed beneath it to provide a guide for determining the location of the channels 80 to be formed. Channels 80 are formed by placing the T-square 46 at a desired location, and removing the gel 76 along a line defined by the T-square parallel to the side border portions 4211, 420. This is best accomplished with the capillary pipette 58, shown in FIGURE 5. The open end 61 is placed against the gel surface 76 at the top of the cutting board 38, just inside the border portion 42b. The pipette is held at an angle of about 20 degrees from the horizontal, pointing in the direction in which it is to be moved along the film 67. The vacuum pump 60 is energized, and the pipette is moved along the film, being guided against the straight edge surface of the T-square 46.

The entire preparation of the starch-gel film is accomplished while avoiding drying the gel, and takes about 10 minutes. As illustrated in FIGURE 12, the channel 78 extends completely through the gel layer 76 so as to isolate the strips 79 from the gel adjacent thereto. This prevents separate samples from spreading to adjacent strips.

After the layer of gel has been channelled into spaced strips of starch-gel on the film 67, the coated film is placed 8 in an electrophoresis cell 85, shown in FIGURE 13. The electrophoresis cell supports the film 67 draped over a horizontal support rod 86, so that opposite ends of the film 67 extend down into a reservoir of electrolyte in a base 87 of the electrophoresis cell. Two electrodes are provided in the cell, one at one end of the strips 79 and the other at the other end of the formed strips, in the container 87. A wick (not shown) is maintained in contact with the gel at each end of the film 67 to hold the film firmly in position and connect the film electrically with electrodes.

A material to be analyzed, such as serum proteins, is applied to each of the spaced test strips 79ae at the top of the electrophoresis cell 85. A starting line may be marked for reference. An electrical current is then applied along the test strips 79 to cause ionized particles of test material to migrate through the starch-gel medium along the the test strips 79a-e. Different particles, due to their different size and mobility in the starch-gel medium, migrate different distances in any allotted time and thereby become separated from other particles of different types.

By way of a specific example, five samples of ten lambda serum are placed at the top of the test strips 79ae adjacent a starting line, which can be applied with a chinamarking pencil. The serum is applied carefully so that the gel is not cut during the application. A spot of reference dye is placed at the starting line, on one edge of the gel, to act as an indication of how far fractions of the test material are migrating. With a Tris-Borate Buffer system by way of example, the serum proteins will migrate from the negative to the positive pole when a direct electric current is applied along the test strips. The positive lead of a power supply is therefore connected to the lower side of the film at the far ends of the channels and the negative lead of the power supply is connected at the end of the film 67, where the channels were not completed, or alternatively, at the starting line on the strips if the electrophoresis cell is constructed to facilitate this. With the present example, only those portions of the strips 79 are being used that extend from the top of the cell 85 to the lower end of the film 67, where the channels 78 extend to the end of the film. With the cell maintained at room temperature, 68 degrees Fahrenheit, a direct current of volts and 4 milliamps are applied to the electrophoresis cell.

The current is applied for approximately 16 to 17 hours, and at the end of this time the reference dye migrates 10 to 11 centimeters from the application point. During this time, different proteins of the serum will migrate to different extents, depending upon their characteristics.

The starch-gel film 67 is then removed from the electrophoresis cell 85 and placed on the cutting board 38, which has been moistened with water to hold the film in position. The film is placed with the starch-gel side up and with the portion that has not been channelled into strips extendlng from the open end 40a of the board. The unchannelled portion of the film is cut away.

The starch-gel film is then placed in a protein staining solution to stain the proteins. The starch-gel sheet is then d e-stained to clear the background, using standard technrques.

By way of specific example, the starch-gel film is immersed in a conventional protein stain for approximately /2 hour. It is then de-stained as follows: A stock solution of methyl alcohol, distilled water and glacial acetic acid in proportions by volume of 5 :5 :1 is prepared. A de staining solution is made by mixing 600 ml. stock solution plus 400 ml. of distilled water. The starch-gel sheet is transferred from the staining solution to the de-staining solution for a period of 2 to 3 hours. After de-staining, only the dye bound to the different fractions of the test material remains.

To further facilitate handling of the starch-gel sheet in the present system, the gel is glycerinated. This step is important to assure that the starch-gel can be handled without damaging the test strips. By way of specific example, the starch-gel sheet is placed in an 8.0% glycerine solution (80% glycerine, 20% water, by weight) heated to 80 degrees centigrade. The starch-gel sheet remains in the solution for one (1) minute, the excess glycerine is then wiped from the sheet and the sheet is left at room temperature for /2 hour. The temperature at which the glycerine is maintained is critical. The temperature must be 80 degrees centigrade, although a variation of not more than :1 degree centigrade may be acceptable. At lower temperatures than 80 degrees centigrade, the glycerine will not penetrate evenly into the gel, even for times of one hour or more. If the temperature is higher than 80 degrees centigrade, air bubbles in the gel explode, or otherwise damage the gel unacceptably. When the glycerinated starch-gel sheet is removed, it becomes plasticized, rendering it resistant, hardened, and better adhered to the film base. As a result, it can be handled and scanned in automatic scanning equipment.

Scanning is accomplished in a conventional recording scanner and integrator that utilizes a light source and a photocell. One suitable analyzer is a model R Spinco Analytrol, sold by Beckman Instruments, Inc., Palo Alto, Calif.

To prepare the starch-gel sheet for analysis, it is again placed on the cutting board 38, this time with the channels 78 parallel with the open end 40a. The channels are aligned with the end 40a of the panel 40, clamped in place by the flat plate 49 of the clamping member 48, and the film is cut along the channels, using the flat plate 49 as a guide. Each cut strip 79 is then separately analyzed by photoelectrically scanning it in an analyzer. This is accomplished as diagrammatically shown in FIGURE 14. The matte strip 70 is placed over the starch-gel strip 79a, with the starch-gel sandwiched between the supporting film 67 and the matte strip 70. The sandwich strip is moved by rolls 90 between a light source 91 and photocell 92 connected with an amplifier 93 that controls a recording pen (not shown), which automatically records on a chart a trace of color density verses distance along the test strip 79a. The area under the curve is then integrated to facilitate measuring concentration of each component of the test material.

In summary, a new system of electrophoresis is provided, using improved apparatus and techniques that facilitate the preparation and use of improved test strips. Thus, it will be apparent that an important aspect of the present system is the provision of a test strip comprised of a starch-gel medium adhered to a film of polyethylene terephthalate, thereby providing a convenient strip with a medium for separating materials to be analyzed into component parts. Such a starch-gel strip can separate 15 to 25 fractions of a protein, whereas paper strips having similar convenience in handling and use, will separate only 5 fractions. As compared with a starch-gel block, a sensitive medium is provided in a new manner which permits the preparation test strips, and use within approximately minutes of the starting time, whereas starch-gel in block form requires at least 8 hours between the making of the gel and the using of it in actual tests. Moreover, in strip form as provided with this invention, the separation of materials can be scanned with an automatic photocell scanner to provide a pattern of density distribution. From this, a relative percent of different fractions of proteins for example can be easily calculated. The preparation and use of such test strips is facilitated by the apparatus and methods described in detail for coating a sheet of plastic film material, providing channels in the sheet, glycerinating the starch-gel to facilitate handling the test strip, and providing a matte strip for facilitating the use of the optically clear base film in scanners constructed for use with diffused light.

While a preferred embodiment of this invention has been described in detail, it will be apparent that various modifications or alterations may be made therein without departing from the spirit and scope of the invention, as set forth in the appended claims.

What is claimed is:

1. In a method of preparing an article for separation analysis of materials, especially biochemical materials, by electrophoresis, the steps comprising:

(a) connecting one end of a sheet of plastic film to a horizontal cylindrical drum,

(b) winding the sheet about the drum,

(c) dipping a portion of the drum across the entire width into a starch-gel medium useful for separation analysis of materials by electrophoresis, with the outer end of the sheet out of the material,

(d) pulling the outer end of the sheet and rolling the drum and sheet through the medium at a substantially constant speed so that the sheet is unwound from the drum and becomes spaced from the drum only above the medium,

(e) whereby the outside surface of the sheet of plastic film comes in contact with the medium while the inside surface does not and the sheet of film material is uniformly coated on one surface only with the medium.

2. The method of claim 1 wherein the sheet is comprised of two separate parts temporarily fastened together, a first part attached at one end to the drum and a second manipulating part removably attached to a free end of the first part and wherein the second part is drawn through the starch-gel medium prior to the first part to remove bubbles that may exist in the medium and is thereafter removed from the first part.

3. The method of claim 1 wherein the starch-gel medium is at a temperature of between 200 and 210 degrees Fahrenheit.

4. The method of claim 1 including the steps of:

(f) forming parallel channels in the starch-gel along a portion of the film to provide spaced strip portions of starch-gel on a single sheet of film,

(g) depositing a material to be analyzed upon the strip portions of the starch-gel,

(h) subjecting the strips to an electric field until particles of the material being analyzed migrate along the strips,

(i) coloring with a stain the material being analyzed in the starch-gel,

(j) glycerinating the starch-gel,

(k) cutting the film into separate strips along the channels, and

(l) scanning a strip photoelectrically to determine the variations in color density along its length.

5. The method of claim 1 including the steps of forming parallel channels in the gel medium along a portion of the film to provide spaced strip portions of gel medium on a single sheet of film.

6. The method of claim 5 wherein the parallel channels are formed by moving a pipette through the layer of gel medium and drawing away portions of the layer with a vacuum.

7. The method of claim 5 including the steps of:

(c) depositing a material to be analyzed upon the strip portions of the gel medium,

(d) subjecting the strips to an electric field until particles of the material being analyzed migrate along the strips,

(e) coloring with a stain the material being analyzed in the gel medium,

(f) glycerinating the gel medium,

'(g) cutting the film into separate strips along the channels, and

(h) scanning a strip photoelectrically to determine the variations in color density along its length.

8. The method of claim 1 including the steps of providing glycerine at a temperature of degrees centigrade placing a starch-gel coated sheet in the glycerine for 1 1 12 approximately one minute and then removing the sheet 3,061,472 10/1962 Brockway 117139.5 and allowing it to dry. 3,303,044 2/ 1967 Fenley 11734 9. The method of claim 8 wherein the glycerine is a solution of 80% glycerine and 20% water, by volume. JOHN MACK, Primary E i References Cited 5 A. C. PRESCOTT, Assistant Examiner UNITED STATES PATENTS US. Cl. XR 343,375 6/1886 Howe 117-115 117 115 13 ;1 5 2,843,540 7/1958 Ressler 204-180 2,962,425 11/1960 Sharpsteen et a1. 204180 10 UNITED STATES PATENT OFFICE (s/ss CERTIFICATE OF C R N Patent No. 3, -97, l- &l Dated Februa 21;, 1970 nvem fl I JOSeph Paksi It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 5, line th, "32a" should be 42a Column 7, line 37, "channel" should be channels Column 7, lines 70-71, "channel 78 extends" should be channels 78 extend Column 10, line 51, "steps should be step A 2: 2;. El-3 :17; 1 '0 n w k L if? v SIGNED AND $EME Amt:

Edward 1!. mad-er, Ir-

Mmmllg 0mm IRMA! I. samrmm, m.

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