David Racusen and Lee White Department of Microbiology and Biochemistry University of Vermont Burlington, 05401
I 1
A Unified Apparatus for Paper and Gel Electrophoresis
Electrophoresis in solid media is a common research tochniquc that has considerable merit in the teaching laboratory as a dircct means of dcmonstrating the ionic properties of amino acids and proteins and for analyzing unknown mixtures of these substances. The apparatus described in this report may be used for either paper electrophoresis or for gel electrophoresis. Especially useful in a feature that allom students to begin or tcrminatc their ovn gel runs without disturbing other runs. Individual gel tubcs arc attachcd to Utubcs forming candy cane-shaped units which simply dip into tho top electrode buffer. The dimensions and operating proccdurcs arc given in detail although they may be chaugcd to suit other needs.
be used including the home-madc, solid-state device described by Davis.' (Warning: Somc power supplies can drlivcr a shock wen after being turned off. Do not touch the electrodes until they have been short circuited or until the output voltage has fallen to zcro as measured by a voltmeter.) The following instructions were used in student experiments. Paper Electrophoresis of Amino Acids2 Moferiols
Filter paper (Whatman No. 1 or 3 1111) about 1 in. wide by about 11 in. long. About 200 rnl of pH 6.3 buffer, pyridine, acetic acid, water (101 0.4/190 v/v) Amino acids (about 0.1 mg/ml) and tracking dye solutions (yellow food color) Procedure
Figure 1 .
The opporotur orronged for poper elecfrophoreril.
Figurc 1 sho~vsthe apparatus as arranged for paper rlectrophorcsis. The ovcrall dimensions wem about 12 in. long X 3L/ain. high X in. wide. All structural mcmbers wcrc Plcxiglas in the following thiclmesses: in.; a central paper case, 3/1a in.; a snugly fitting lid, support, in.; and two electrode boxcs, in. Strips of in. Plexiglas w r c cementcd to the inner walls of the case forming a slot into which the paper support would slide freely. Power connections were made by clamping onto the stainless steel lugs which projected through in. holes. Two extra ' / I in. holes were located in thc floor of thc casc. One of the electrode boxes was built with elevated side and back walls to serve as a support for the other box when they were stacked for gel electrophoresis. The lugs then protruded through the extra holes noted above (Fig. 2). The electrodes were of platinum wire held firmly to the floor of the boxcs by small Plexiglas blocks cemented in place. Any power supply providing 150 V dc could Vermont Agricultural Experiment Station Journal Series No. 263. D - ~ v l sB., , Ann. N. Y. Acad. Sci., 121, 404 (1964). E.,, A N D ZW:IG,G., "A Manual of 2 R ~ o Ii., ~ ~I ~ ,U R R U M Paper Chromatography and Paper Electrophoresis" (2nd ed.), Academic Press, Inc., New Yark, 1958.
Apply the amino acid solutions as a streak on the folded midpoint of the paper. To the dried streak apply a small spot of dye solution. Carefully soak the paper with pH 6 3 buffer so that the origin is nut displaced by cnpillnrity. Blot t o remove excess buffer and drape the strip tautly over the plastic paper support so that the origin is in the center and the ends dip into the electrode hoxes. (The apparatus will accommodate three or four such strips.) With Lhe power supply not plugged in, connect the clamps to the electrodes and cover the entire ehamber. Activate the power source. At 150 V the yellow tracking dye will move about 2 in. in an hour at which time the run is eompleled. Turn off the power supply and remove the paper strip, discarding the drenched pieces at the ends, and dry in the oven or hood. Ileleet the amino acids with 1% ninhydrin in acetone a t 60°C for 10 min or at room temperature overnight.
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Figure 2.
The apparatus arranged for gel elecfrophoresir.
Volume 49, Number 6, June 1972
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439
u-tube filled with gel razor slit
Figure 3.
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50% Glycerol containing about 0.1 ml of 0.04Y0 bromphenol blue per 10 ml of solution 0.01% Amido blackin 7% acetic acid 7% Acetic acid 4 Glass tubes (5 mm i d . X 65 mm long)
tygon hose
Procedure
protein sample in glycerol
Mix 0.5 ml of HzO, 0.5 ml of solution A, and 1ml of solution C ; then add 2 ml of ammonium persulfate. Warm t o about 35%, blend thoroughly, and immediately pipet 1 ml into each of 4 tuhes set vertically in leak-proof rubber bases. Layer carefully with a t least in. of H20 using a fine-tipped pipet. Allow to stand until the mixture has gelled (about 10 min). Shake out the water layer and into each tube pipet about 0.1 ml of a sample mixture which has been made to 10% glycerol and indicator. Fit short lengths of tygon hose over the top end of the tubes and carefully overlay the protein solution with electrode huffer, lilling t o the top of the hose. While holding each tube vertically, slide a gel-filled U-tube into the hose so that no air is trapped between liquid and gel. A small razor slit in the hose allows air and some buffer t o be displaced during this connection (Fig. 3). (The U-tubes contain the same gel described above and may he used indefinitely if stored in the electrode huffer.) Hook the free end of the U-tube onto the top buffer box so that a bubblefree connection is made between the top and bottom buffers. Plug in the power supply (150 V dc) and in a few minutes observe the refractile protein zones as they move int,o the gel behind the bromphenol blue front. The run may he allowed to proceed until the front is near the bottom or far about 45 min. Unplug the power supply and remove the tube assemblies. (Re-plug if other tubes are being run.) Remove the U-tube and return it t o its buffer bath. Rim the gels under tap water with a. long needle until they slide out freely. Stain one pair of gels in pH 7 buffer, guaiacol, and peroxide, and sketch the position of the brown pemxidase zones which appear in a few minutes. The other pair oi gels may he stained in amida black overnight and destained by repeated changes of 7% acetic aoid over several days' time. All staining should be commenced promptly before the protein zones have a chance t o diffuse.
~d U-tube ~ g for ~ g rd electrophorerir. n ~ ~ t
Discontinuous Electrophoresis of Protein on Polyacrylamide Gel' Materials Leaf or other protein solution (-0.5 mg/ml) in 0.025 M phosphate pH 7 Solution A (st,ahle a t 4 T ) : 1 N HCl, 48 ml; 2-amino-Whydroxymethyl)-1,,7-prop~nediol (Tris), 36.6 g; N,N,N',N'Tetramethylenediamine, 0.46 ml; HIO t o 100 ml. Solrtdion C (20% acrylamide and 0.8% bisaerylamide) (stable a t 4°C): 0.14% Ammonium persulfate (catalyst) (stable a t 4°C)
~ l e c & d e buffer (stable at 4°C): tris, 0.60 g ; glycine, 2.88 g; H 2 0 t o 1000 ml Peroxidase stain (as needed): 0.02.i M phosphate pH 7, 7 ml; 0.3% H2Oo 1ml; 1.5% guaiacol, 2 ml
440 / Journal of Chemical Education
