Jack Tibbs
Medical Sciences institute University of Dundee Dundee D D I ~ H N United , Kingdom
II
Simple and Safe Electrophoresis at LOW and High Voltage Gradients
The author was faced with the task of designing and providing, on a limited budget, electrophoresis equipment suitable for student use in studying amino acids, peptides, and proteins. It had to be simple, for it was necessary to accommodate substantial numbers of students a t any one time, and also as versatile as possible. Electrophoresis times had to be short, but from the point of view of student safety, applied voltages had also to be low. The problem is one which must have faced many teachers of biochemistry, and in the past we have usually avoided it by having a very limited number of carefully supervised unit experiments. Considerations Involved in Designing Suitable Equipment Much of the apparatus commercially available for cellulose acetate strip electrophoresis is not really suitable for class work. Tanks are often larger and strips longer than necessary. This can give rise to equilibration problems and strips must be supported in the center when they are positioned in the tank. Such strips and tanks are not particularly easy for inexperienced students to manipulate. When proteins are separated on cellulose acetate, maximum resolution is usually obtained on about 3-4 cm of the strip. Long strips (16-20 cm) are not required and if used, applied voltages or run times are unnecessarily high. Small tanks, making use of shorter lengths of cellulose acetate score on all counts. They are easier to handle and store in quantity. No equilibration period seems to be necessary, and first-class separations, spread over 3-4 cm, ORIGIN
of serum proteins have been achieved in 40 min using an applied voltage of 100 V and a voltage gradient of about 15 V/cm (Fig. 1).Such separations are certainly the equal of anv obtained in larner tanks. ~ h e s econsideratio& are probably not new, hut making use of the high resolving Dower of commerciallv available layers of silica Gel bonded to an inert polyester backing ("Chromogram" Type 6061, manufactured by Eastman) may be extended to include separations of amino acids and peptides. For several years 5 X 5 cm squares of this material (cut with scissors from larger sheets) have been used by classes in this Department for carrying out chromatographic separations, and providing the applied spots are small, separations are rapid and resolution often spectacular. Squares of this size are now being used to combine electrophoresis at high potential gradients (about 20 V/cm but with an overall voltage of only 100 V) with chromatography to separate peptides from each other and to ~ r o v i d esuccessful rotei in "finnemrints" (Fin. 2). The complete two dimensional operacon is complete within one hour. The apparatus is the same as that employed for the cellulose acetate work and without much difficulty could probably he adapted to gel electrophoresis. Construction of Apparatus With the above points in mind, the design of the apparatus becomes almost obvious (Fig. 3). Constructed of Perspex,l i t contains the three usual partitions, the center partition isolating the two electrode compartments from each other being 2-3 mm lower than the two others. These two outer partitions, perforated along their length below the buffer level, divide each electrode. compartment into two regions; the inner region contains the platinum electrode while the electrophoretic support material makes 1 Trade name for methyl methacrylate polymer.
t
Electrophore~i~ First dimension
Figure 1. Cellulose acetate strip electrophoresis of four samples of human serum. Conditions were 40 min at 100 V in pH 8.6 barbitone buffer. a. Normal serum; b. serum from patient with immunoglobulin G myeloma; c, serum exhibiting polyclonal gammopathy (general increase in immunoglobulins): d. serum with abnormal immunoglobulin M(Wa1denstroms macroglobulin).
862 /Journal of Chemical Education
-Chrom~lto(lraphy Second dimension Figure 2. Peptide map from tryptic digest of sheep globin. Direction1 involved electrophore5is at p H 6.5 in pyridine-acetic acid-water buffer at 100 V for 35 min. Direction 2 involved ascending chromatography in butan-1-01-acetic acid-water (4:l:l. by vol).
serum separated quite clearly into five hands, the alhumin hand travelling ahout 30 mm (Fig. 1). Of the usual protein stains we have chosen nigrosin (0.01% in 2% v/v aqueous acetic acid) for qualitative class work. At this concentration staining.is quick and excess stain is s~eedily . removed by rinsing in tap water to leave a pattern with a high degree of contrast.
Figure 3. Construction details of electrophoresis tank. The lid and the holes below buffer level in the two outer partitions have been omitted from the diagram. A. Partitions. 75 mm X 32 mm X 5 mm. 8 , partition, 75 mm X 30 mm X 5 mm: C, base, 67 mm X 75 mm X 6 mm (lid is same size): D, sides and end pieces, 75 mm X 50 mm X 6 mm: E, holes lor electrode terminal: F, platinum wire electrodes. 110 mm.
contact with the huffer in the outer region. Internally, the tank is 50 mm high X 63 mm wide (in the direction of migration) X 75 mm long (in the direction of the electrode wires). The platinum electrodes run the length of the tank and are anchored to the base by small blocks of Perspex which have been grwved on the underside to allow the wire to pass through. After the Perspex had been cut to size and the edges milled to the correct length, the author assembled 35 of these tanks in a few hours, and hy using a simple distribution box, 10 of these tanks may be plugged into one power pack. Cellulose Acetate Strip Electrophoresis The tank accommodates two 80 x 25 mm strips. The latter, after soaking in huffer in the usual way, are placed across the two outer partitions so that their ends dip into the outer buffer compartments and are held in place by small pads of 6 mm thick foam rubber inserted in the 6 mm space between the sides of the tank and the outer partitions. The strips clear the center partition. In order t o obtain maximum potential gradient for a given applied voltage the huffer levels should he as high as possible. Using a glass capillary the sample is now applied to the strip as a narrow hand, and for most proteins this will he well towards the cathode end. We have found this method of application to he satisfactory and after a trial run most students are ahle to apply samples fairly evenly as a band about 1-2 mm thick, especially if the protein solution is made visible by adding a little hromphenol blue. If the sample is serum the dye, of course, hinds to the albumin hand and makes it easy to follow the subsequent electrophoresis. It is probably an advantage to place a piece of damp filter paper over the tank before positioning the lid. In 40 min a t 100 V, using pH 8.6 harbitone buffer, human
Silica Gel Thin Layer Electrophoresis For peptide mapping the sample (0.2-0.4 PI, 10-20 fig of peptide) is applied by means of a glass capillary to a suitable point on the gel square to give a spot no more than 2 mm in diameter. After drying the square is impregnated with pyridine-acetate electrophoresis huffer. This is an unpleasant huffer to handle but the impregnation is easily and very satisfactorily carried out by pressing the square, face down, on to a piece of filter paper which has been wetted with buffer and the surplus removed. Smudging of the spot has not proved t o be any trouble and especially when large numbers of students are involved, this procedure is infinitely preferable to spraying. Fume cupboards are desirable but by no means essential. The square is now placed face down and supported by the two outer partitions so that the anode and cathode edges of the Silica Gel overlap the edges of the rectangular filter-paper wicks which have been folded over the tops of these two partitions. Toese wicks, which dip into the outer compartments and run the length of the tank, have been made out of Whatman No. 1 filter paper. One thickness of this introduces ample huffer on t o the thin layer. It may even he an improvement to use a thinner paper. The tank lid is placed in position and the current is switched on. Using pH 6.5 pyridine-acetic acid-water (25:1:225, by vol) at 100 V for 35 min, the peptides in human globin tryptic digests spread over about 40 mm (the absence of overheating was confirmed by the fact that during the run the current remained steady a t about 4 mA). After electrophoresis the square is dried and ascending chromatography or electrophoresis carried out in the second dimension. The solvent is removed and the spots located by heating after using one of the many ninhydrin dips to give results such as those in Figure 2. The drying and heating stages are best carried out and controlled using a hair dryer. Conclusions By exploiting the high resolving power of cellulose acetate and manufactured Silica Gel layers it is possible, using very simple electrophoresis equipment, to carry out sophisticated separations rapidly and safely with large numbers of students. The rapidity means that several separations can be carried out in one class session. In addition to peptide studies, the Silica Gel technique gives first-class results with amino acids and amino acid derivatives and can doubtless be extended to nucleotides and nucleotide mapping. I t should also he applicable to diagonal electrophoresis.
Volume 50, Number 12, December 1973
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