These proportions are at least as characteristic of each species as the structure and proportion of blood cells. Moreover, certain diseases cause marked changes in the serum proteins, so that the electrophoresis apparatus has become a useful id in diagnosis. Graphs of protein concentration, known as phrenograms, have so far been drawn for only a few diseases of a few species, however, and phrenograms of even healthy animals vary somewhat depending on the design if the apparatus and the technique of the observer. As a result it is customary each, experimenter to construct a "normal" phrenogram by analyzing the serum of a number of healthy animals. Electrophoresis equipment is not readily available commercially but it can be devised by an amateur; blood can be obtained from veterinarians or slaughterhouses.

Two basic schemes have been devised for separating proteins by electrophoresis. In one an electric current is passed directly through the solution to be analyzed. The electrified molecules simply migrate toward one pole or the other depending on their size and the intensity of their charge. If the container is long in relation to its width and thickness- a glass tube, for example-the migrating substances concentrate in zones of differing refractive index and can be observed indirectly by measuring the optical refraction of each zone. This technique, known as free electrophoresis, was pioneered by the Swedish biochemist Arne Tiselius [see "Electrophoresis," by George W. Gray; SCIENTIFIC AMERICAN, December, 1951]. In the other scheme, known as zone electrophoresis, the electrified materials concentrate in zones along a strip of filter paper that carries an electric current of a few milliamperes. In the case of proteins the zones are dyed and subsequently evaluated by measuring the amount of light transmitted by each zone.

Richard LaFond of Monson, Mass., makes a hobby of zone electrophoresis. "Human blood serum," he writes, "is resolved into five zones of protein called albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin. Similar proteins are found in the serum of other animals, but the proportions differ in each species. The analysis requires only a single drop of serum. The essential apparatus consists of (1) an electrophoresis cell that separates the proteins into zones on a strip of filter paper, (2) a bath of dye for staining the zones and (3) a photometer for measuring the opacity of the dyed proteins. All the essential materials are available from hardware stores and dealers in laboratory supplies.

"The electrophoresis cell consists of a pair of plastic containers fitted with plastic baffles, a pair of clamped glass plates for supporting the filter paper, a pair of electrodes, each surrounded by a test tube that has a hole in the bottom, and a supply of direct current at 500 volts [see illustration]. I could not locate plastic containers of the desired size, so I made a pair from sheet Plexiglas an eighth of an inch thick. By collecting the sawdust manufactured when I cut the parts of the containers from the sheet and mixing it with acetone, I made an adhesive, of the consistency of light sirup, with which to cement the edges of the assembled containers. I also cemented a baffle in each container as shown in the illustration. The completed vessels were 16.5 centimeters long, 13 centimeters high and 12.5 centimeters wide; in operation they held 1,400 milliliters of solution. The two test tubes used to support the electrodes were 12.5 centimeters long and 1.5 centimeters in diameter. To make a hole in the bottom of each test tube I heated a small spot until the glass softened at the center and then I blew into the open end. The ragged film of glass around the blown-out hole was broken off and the edges were fire-polished by returning the glass to the flame. The electrodes were made by soldering a strip of platinum foil three millimeters wide and four centimeters long to each of two pieces of bare No. 10 copper wire about eight centimeters long. The wires were then pushed through rubber stoppers that fitted the test tubes. A groove cut in the side of each stopper equalized the air pressure within the tube. Electrodes made of carbon welding rod can be substituted for the foil, but platinum is preferred because it is more inert and can be cleaned easily by being heated to redness in a gas flame.

"The serum to be analyzed is applied in a thin line across a strip of filter paper that has been moistened with the solution in the containers and blotted almost dry. The paper is clamped between glass plates supported by the ends of the containers, as illustrated. The containers serve as chemically stable terminals through which the ends of the paper strip are connected to the power supply. The reason for this elaborate setup is that reactions always occur at the point where metal electrodes make contact with a solution that functions as an electrolyte. If the electrodes were connected directly to the paper strip, the unwanted products of these reactions would migrate into the strip and interfere with the migration of the serum proteins. It is to prevent this contamination and the resulting interference with the protein separations that the platinum electrodes are placed in the test tubes, which act as baffles. As a further precaution the tubes are placed in the solution behind plastic baffles, which also help to confine the unwanted substances. Finally, because the ends of the paper strip dip into the compartment of the container that holds a large amount of electrolyte, such unwanted substances as may form there are highly diluted.

"The electrolyte is distilled water in which a chemical is dissolved that forms positive and negative ions. The ions act as carriers of current. The negatively charged ions migrate to the anode, where they give up electrons. The positively charged ions accept electrons from the cathode. The unwanted chemical products are the result of this exchange.

Some of the products are liberated in the form of gases and bubble out of the solution harmlessly. Others, in the form of salts, are trapped in the system of baffles. A substance should be selected for the electrolyte that supplies ions to the solution at the rate at which they are removed by electrochemical reaction. The electrolyte should also be as dilute as possible so that the ionizing substance will not hamper the migration of the proteins. A .05 molar solution of sodium diethylbarbiturate works well. It is made by dissolving 62.2 grams of sodium diethylbarbiturate in 1,500 milliliters of distilled water. The electrolyte must be adjusted so that it is slightly alkaline-to a pH of 8.6. I borrowed a pH meter to adjust my electrolyte, but the alkalinity can also be tested with paper strips treated with phenolphthalein. Changes in the color of the moistened strips serve as an index of pH. The strips are inexpensive and come in a vial with a calibrated color chart. The pH of the electrolyte is altered by adding either dilute hydrochloric acid or sodium hydroxide (lye) as required.

"The completed cell is connected to a source of direct current capable of supplying about 20 milliamperes at a potential that can be varied from 50 to 500 volts as required. The power supply unit in many radio sets will serve adequately. Units from discarded radios can often be bought for a dollar or so from service shops. The output voltages from these units can be controlled by a continuously variable power transformer, such as a Variac. I built my unit from standard components and equipped it with a voltmeter and a milliammeter so that a record could be made of the current and voltage used during each experiment [see Figure 2],

"To prepare serum for analysis, pipette 10 milliliters of whole blood into a conical centrifuge tube and centrifuge it at 3,000 revolutions per minute for 10 minutes. The centrifuge consists of a horizontal arm about 20 centimeters long that supports a pair of hinged fixtures at the ends into which the centrifuge tubes are inserted. The center of the horizontal arm is fixed to the upper end of a revolving shaft that is mounted in bearings at the bottom and driven through gears by either a hand crank or a motor. The hinged fixtures enable the bottoms of the test tubes to swing outward as the machine comes up to speed. The machine is balanced by partly filling the second tube with water. Centrifuge tubes can be bought at drugstores and the remainder of the apparatus can be improvised from scrap materials. After 10 minutes the solid part of the centrifuged blood will have largely collected in the pointed end of the centrifuge tube, leaving the straw-colored serum at the top. The serum is decanted into a clean tube and centrifuged an additional five minutes at 3,000 r.p.m. The upper half of the serum is then decanted into a vial and stored in a refrigerator at a temperature of 45 degrees Fahrenheit. "The cell is prepared for an experimental run by first cutting a strip four centimeters wide and 10 centimeters long from a sheet of Whatman No. 3 filter paper and drawing a pencil line squarely across the strip .5 centimeter from one end. (Be careful not to scratch the paper with the pencil.) The strip is then saturated with electrolyte and blotted as dry as possible between two sheets of filter paper.

"A thin line of serum must now be applied along the pencil line. Fair results can be achieved by taking up a few drops of serum in a pipette and drawing the tip across the strip. But it is better to use a special applicator: a pair of fine, closely spaced parallel wires five centimeters long stretched between the tines of a short fork.

"To make the fork, bend a 25-centimeter length of heavy iron wire double at the middle like a hairpin and clamp the free ends in a vise five centimeters below the loop. Insert a nail through the loop for a handle and twist the loop several times. Remove the wire from the vise and bend the free ends to form a pair of tines one centimeter long spaced five centimeters apart. The fine wires are made from the E-string of a violin. Fold an 11-centimeter length to make a double strand and solder the ends so that the wires stretch between the tines. Be sure not to solder the fine wires together except at the tines. When the solder cools, insert a razor blade between the strands at the ends where they are attached to the tines and spread them just enough to make a thin slit.

"Clean the completed applicator thoroughly. Then place a single drop of serum on the wires at the center. Capillary attraction will make the serum flow evenly across the slit. Transfer the serum to the filter paper by pressing the wires gently against the pencil mark. A straight, sharp line is wanted; getting it can be simplified if the serum is diluted with one part phenol red to two parts serum. The specimen will then appear as a pink line across the paper and any smudging will be evident. Phenol red does not react with proteins and migrates out of the paper when current is applied to the cell.

"The inner faces of the glass plates are coated lightly with silicone grease, which can be bought in tubes at radio supply stores. The paper is sandwiched between the two coated plates. The assembly is secured by clamps as shown, greased lightly along the sides to prevent evaporation add placed on the ends of the containers. Check to see that the level of the electrolyte in the two containers is the same, or it will flow from the higher to the lower container through the filter paper and interfere with the migration of the proteins.

"Connect the power supply to the cell, with the negative lead attached to the terminal at the specimen end of the paper strip. Switch the current on and adjust the potential to 300 volts. If the electrolyte is at the specified concentration and pH, the cell will draw about two milliamperes at this voltage. Switch off the current after two hours and tear off the dangling ends of the paper as close as possible to the glass plates. This prevents excess electrolyte in the discarded ends from flowing into the protected portion of the paper and smearing the zones. Release the clamps and remove the strip carefully. Place it on a sheet of dry filter paper and, keeping it horizontal, put it in an oven at about 250 degrees F. for 30 minutes. The heat binds the proteins to the paper and increases their affinity for dye.

"The proteins are then stained by immersing the strip for 16 hours in a solution of 50 milliliters of glacial acetic acid, 50 grams of zinc sulfate and .1 gram of bromophenol blue dissolved in distilled water to make one liter. This will color the whole strip deep blue. The paper is then rinsed twice for five minutes each time in a 4 per cent solution (by volume) of acetic acid, with fresh solution being used for each rinse. The protein zones should now appear as a series of blue transverse bands of varying density against a background of pure white. Next the strip is immersed for two minutes in a fixing solution made by dissolving 20 grams of sodium acetate and 100 milliliters of glacial acetic acid in distilled water to make one liter. Then the paper is blotted, dried in an oven for five minutes at 250 degrees, placed close to a tuft of cotton saturated with household ammonia and covered with a mixing bowl for 10 minutes.

"The significance of the electrophoresis technique becomes apparent when the density of the zones is measured and plotted as a graph. The measurement is made by means of a densitometer, a photocell that indicates the intensity of light transmitted by the stained proteins through a slit moved along the strip. A densitometer can be improvised from an ordinary exposure meter of the type used by photographers, a sheet of glass set in a frame and some cardboard. Cut a piece of cardboard large enough to mask the photocell aperture, and with a razor blade cut a straight slit in the center of the mask about two centimeters long and one millimeter wide. Fasten the mask over the photocell with adhesive tape. Then, with a pencil, draw a series of index marks precisely one millimeter apart along the full length of the prepared electrophoresis strip at one edge. Center the indexed strip on the framed glass, with the bottom face of the strip in contact with the glass, and attach the ends to the glass with adhesive tape. Clamp one edge of the frame to the edge of a desk or table so that the glass extends out from the support, with the paper on top. Place the slit of the exposure meter squarely over the most intensely dyed zone and position a 100-watt incandescent lamp below the glass at a distance such that the pointer of the exposure meter is deflected slightly by the light that comes up through the zone. Then shift the slit along the strip, one millimeter at a time, and tabulate the deflections for each interval. The accuracy of the measurement can be improved by taping a ruler to the glass so that the slit can be kept at right angles to the edge of the strip and shifted parallel to it. A densitometer that is of higher sensitivity and more convenient to use can be assembled by connecting a silicon solar cell, such as the International Rectifier Type S1020E8-PL, to a milliammeter calibrated to read 30 milliamperes at full scale.

"A graph of protein density in rectangular coordinates is made by plotting the tabulated meter readings against the length of the strip (in millimeters). The concentration of proteins in human blood from a healthy person should resemble the accompanying graph [Figure 3]. To compute the relative proportions of the proteins, measure the area under each of the five humps in the curve and calculate the percentage of the total accounted for by each.

"Departures from this normal graph have been identified with a variety of diseases. Prolonged malnutrition and diseases of the liver are associated with abnormally low albumin. Abnormally high levels of alpha-2 globulin, such as that indicated in the second strip from the top in the accompanying illustration [above], can indicate inflammation or tissue destruction. Antibodies are included in the gamma globulin zone; low values may indicate poor natural resistance to disease. Zone electrophoresis is particularly helpful in diagnosing agammaglobulinemia, a condition characterized by the absence of gamma globulin.

"Interesting differences in the proportions of serum proteins have been tabulated for a few animals. In the case of human blood, the level of albumin normally ranges from 47 to 71 per cent of the total serum proteins, with a mean value of 59.2 per cent, whereas in mature horses it ranges from 50 to 55 per cent, with a mean value of 51.8 per cent. The corresponding mean values of alpha globulin reported in human serum and horse serum are 12.5 per cent and 17.1 per cent, for beta globulin, 11.4 and 16 per cent, and for gamma globulin, 15.1 per cent and 16.6 per cent.

"Normal values have been tentatively established for cows, pigs, dogs, rats, mice and alligators. In general the graphs appear to differ increasingly from those of human serum as animals at progressively lower levels of evolution are analyzed."

Bibliography

MANUAL OF PAPER CHROMATOGRAPHY AND PAPER ELECTROPHORESIS. Richard J. Block, Emmett L. Durrum and Gunter Zweig. Academic Press Inc., 1958.

THE SCIENTIFIC AMERICAN BOOK OF PROJECTS FOR THE AMATEUR SCIENTIST. C. L. Stong. Simon and Schuster, lnc., 1960.

The Society for Amateur Scientists (SAS) is a nonprofit research and educational organization dedicated to helping people enrich their lives by following their passion to take part in scientific adventures of all kinds.