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FILTER PAPER ELECTROPHORESIS A Biophysical Laboratory Experiment SISTER MARY MARTINETTE, B.V.M. Mundelein College for Women, Chicago, Illinois
B I O C H E M I ~ A L experiments showing the separation of the several proteins in blood serum have been successful to only a limited degree. Many biochemists have felt that the fractionations are incomplete and that it is questionable whether definite substances are isolated. The ammonium sulfate method of fractional precipitation which is widely used is only moderately satisfactory. An interesting yet very simple technique has been developed using filter paper electrophoresis. Wieland and co-workers (1, t)in Germany, Durrum (3) in this country, and Tiselius and co-workers (4, 5) in Sweden, each independently published reports based on the same theory. The method is a qualitative one. A high degree of precision is observed when identifying the individual
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Apparatus for Filter Paper QSctroph0-i.
The filter paper strip .i shown clamped between glass plates which are resting on the Lips of the electrode vessela. Two albumin spots are shown migrating from the line of origin toward the anode.
components but it is doubtful that the method as now worked out would give accurate quantitative data. One of the decided advantages of the new technique is the fact that both minute quantities of amino acid mix-
tures and normal quantities of protein constituents may be separated (3). The method is one in which an electrical potential is applied across the ends of a strip of filter paper which has been saturated with either buffer or some other electrolyte solution. At narrowly circumscribed points mixtures of amino acids, peptides, or proteins may be put on the paper. A current is then applied and allowed to run for a period of from three t o 15 hours. At the end of this time the positions to which the components have migrated is determined by spraying the paper with, or dipping it into, a developer. Durmm's method (5) employs the bromphenol blue dye and is extremely sensitive. The author has used the method t o demonstrate both electrophoresis and the separation of proteins in blood plasma. The experiment fits well into the physical chemistry laboratory schedule for the second semester. Quite possibly it could also be used in a biochemistry laboratory course. The proposed experiment outline is as follows: Purpose: The separation of blood serum proteins. Theory: The electrolytic separation of colloids is one of several methods of separation. Colloidal particles migrate in an electric field but are extremely sensitive to changes in pH. The charge carried by a protein particle is governed by the particular property which is effective in the given environment. In relatively acid solutions protein molecules, being amphoteric, behave as bases and will combine with the free protons, acquiring a positive charge. In alkaline solutions they mill transfer protons to the available hydroxyl ions leaving negatively charged particles. These dharacteristics are the basis upon which electrical separations may be made. When the migration is allowed to proreed through a moist filter paper medium there is sufficient differencein migration rates to bring about a separation of the individual colloidal particles. Apparatus: Source of 110 or 220 volts d. r., two carbon electrodes, filter paper strips (Schleicher and Schuell #547 or Whatman 3 mm.), two beakers (500 or 1000 ml.), glass plates (rectangnlar, approximately 4 X 7 in.) and two clamps. Procedure: (1) Partially fill two beakers with a buffer solution of pH approximately 7.5. (2) Cut two strips of fdter paper of convenient size to span the distance covered by the rectangular glass plates and to dip into each buffer solution.
(3) Draw a pencil line down the center of one strip of the paper a t the point which will be equidistant from the electrodes. (4) Rub a thin film of silicone grease on one side of each of the two glass plates. (5) Dip the lined paper strip into the buffer solution and partially dry it by blotting it with the second paper strip. (6) Set the moistened paper strip on what will be the lower glass plate and line the sides of the paper with silicone grease in order to avoid evaporation during electrophoresis. (7) Withdraw a sample of serum (0.005 t o 0.010 ml.)and apply i t a t any point on the previously drawn line. A small amount of bromphenol blue added to the serum aids in following the migration of the albumin which binds this dye. (8) Place the upper glass plate firmly over the paper and clamp tightly with light clamps (Figure 1). (9) Place the plates on the lips of the electrode vessels with the protruding ends of the paper dipping into the buffer in the vessels. (10) Apply a voltage of 110 to 220 volts d. c. If a high voltage is used regulate the resistance to avoid heating the paper strips. (11) Allow the experiment to run from three to 15 hours. Following the separation remove the paper carefully to avoid smearing; dip it into a one per cent solution of bromphenol blue in ethyl alcohol which has been saturated with mercuric chloride. After two to fiveminutes pour off the dye solution, which may be used repeatedly, and wash the paper with water which has been slightly acidified with acetic acid. Dry the paper and, if necessary, pass the dried paper through ammonia vapor to bring out the protein spots. Albumin a, a2,0, and y amear as a seanence of svots leading out from the ~ e n c i l I& (Figure 2j. The author found that migration is apparent in a matter of 20 minutes a t a voltage of 130 v. It is quite that wide variety of 'Iter papers may he used. As with a transference nnmber exoeriment, the experiment can easily be set up and let &n while the
students proceed with other work. It requires no watching during the course of the electrophoresis. If samples of normal serum and of pathological serum are available the resulting deviations in pattern are very interesting (Figure 2) and serve to instruct the students
A Normd Serum (Upper) C o n k t d vith T h Patholcqir.1 Sea: Cir.ho.i.. Nephrmie ond Mydomm in D-ending Cder
Figure 2.
as to the practical use of the method in medical diagnosis. Many uses for the method have been proposed. Clinical applications appear to be particularly numerous (6). Attention to detail and careful technique are essential to good results and the students find the experiment interesting, challenging, and instructive. LITERATURE CITED (1) WIELIIND, T.,
AND
F. FISHER, Ndulwi88mchaften,
35, 29
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(2) WIELAND. T., AND L. WIRTH,Angew. Chem., 62,473 (1950). (3) D m m u ~E. , L., J. Am. C h a . Soc., 72,2943 (1950). (4) C ~ M E R H., D., AND A. TISELIUS,B i o c h a . z., 320, 273 (1950). (5) KUNKEL,H. G., AND A. TISELIUS,J . @en. Physiol., 35, 89 1 10~1) ,*""*,.
(6) Seminal; Spring issue, 20-3 (1953).
