Disc electrophoresis

John M. Brewer

and Raymond B. Ashworth University of Georgia

Disc Electrophoresis

Athens 30601

Disc electrophoresis involves first concentrating protcin or nucleic acid samples into very thin layers using a discontinuous voltage gradient, and then electrophoresing them on a column of polyacrylamide gel. The concentration step allows electrophoresis from starting zones only 1-100 microns thick, which produces the enhanced resolution characteristic of the technique. The acrylamide gel stabilizes the zones of protein formed during electrophoresis, so that convection (the protein zones are denser than the solvent) does not occur. By varying the concentration of acrylamide and methylene bisacrylamide (the source of cross-links) in the gels, fractionation of the proteins on the basis of their size, as well as their charge, can be achieved, because larger molecules will encounter greater resistance in moving through the gel network than smaller ones. To understand why the protein concentrates, consider the "standard" system (Fig. 1). negative electrode

L:rzr

,f

(pH 8.3)

t r i s * =0.0025M=tri \gIy=ine-=O.O073M glycineo=0.04M

u--, \0

Lower gel (pH 8.9)

triP.0.315~ chloride-=0.0 M

r--Electrode buffer

Figure 1.

This reduces the effective mobility of glycine further, to very much lower than that of chloride. The chloride and glycine solutions are electrically in series, and the current-the movement of ions-must be the same throughout the system. So the ions must move with the same velocity. For this to happen, the voltage must be considerably higher in the glycine region than in that containing chloride. This discontinuity in voltage gradient (the voltage divided by the distance) effectively prevents diffusion at the interface between the two solutions. A chloride ion moving into the glycine region is accelerated out again by the higher voltage gradient. A glycine anion attempting to diffuse into the chloride solution finds itself in a region of much lower voltage, and slows down until the rest of the glycine catches up. If proteins are also present whose mobilities are between that of glycine a t pH 8.3 and chloride, these will "sandwich" themselves between the two solutions to form their own zone of intermediate voltage gradient. Diffusion a t both ends of the protein zone and between discs of protein of different mobilities within the protein zone will be again restricted, for the same reason that diffusion between the glycine and chloride solutions is restricted. Note that in this zone, the only anions present will be the proteins. Because of the low net charge to mass ratio of most proteins, the proteins must concentrate until each can carry, in its sub-zone, as much currrent as the chloride and glycine solutions. Thus the protein zone will consist of thin discs of all the proteins with the right range of mobilities, arranged in order of increasing mobility. This concentration step occurs in the "upper gel." These discs, being immediately adjacent, are of no analytical use. They must now he separated by ordinary electrophoresis. This is done in the "lower gel." As mentioned before, the average net charge, and consequently the effective mobility, of a solution of glycine will vary with pH. Note that when tris is used as the counterion as in the standard system the conjugate hase is uncharged. So when the glycine zone migrates into a region where the concentration of tris hase is high, as is the case in the lower gel, the pH will rise (to 9.5 in the standard system), and the concentration of glycinate anion will increase. This will produce an increase in effective mobility of glycine (and consequently a decrease in voltage gradient) in the lower gel to where it will be higher than the effective mobility of the proteins, though not of course of the chloride, which it cannot surpass. The glycine front will then pass through the protein zone, and the proteins will now he electrophoresing in the glycine solution a t pH 9.5. This will occur even though the net charge, and hence the effective mobility, of the proteins will also increase.

positive electrode

Ionic comporitionof the "standard" system.

When a voltage is applied across the solution, the ions move, positive ones to the negative pole and vice versa. The movement of the ions constitutes the current. The mobility or rate of migration of glycinate anion is lower than that of chloride anion under identical conditions. I n addition, at pH 8.3, the average net charge on the glycine is 1/30 that on the chloride (which is one).

Volume 46, Number I, January 1969

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If nucleic acids or very highly charged (acidic) proteins are present, these may still have mobilities exceeding that of glycine a t pH 9.5. These will then stay immediately ahead of the glycine solution, and will not be visibly separated. The "tracking dye" (see below) has a mobility which is independent of pH in this region and is intermediate between chloride and glycine. It therefore remains at the glycine front and marks its progress. If the lower gel pH is progressively lowered, so that the concentration of tris base is decreased while maintaining the same chloride concentration, the "running pH" in the glycine region will decrease so that the concentration of glycinate anion and hence the effective mobility of the glycine will drop. The voltage gradient will increase, and so the proteins will migrate relatively faster in the lower pH glycine until at some lower gel pH (about 7.5-8.0 in the standard system, depending on minor variations in tris and glycine concentrations and pH) all the proteins will have a higher mobility than the glycine and will migrate just ahead of the glycine front. The concentration of tris base is naturally made very low in the upper gel (pH 6.9), so that the protein will "stack" and migrate at the glycine front until it reaches the lower gel. Phosphate has a much greater mobility than the glycine, so it is used instead of chloride because of its superior buffering capacity at that pH (originally, a pH 6.9 tris-chloride buffer was used). The buffer used in the lower dish is the tris-glycine buffer. It simply serves to make electrical contact between the gels and the positive electrode. A tris-HC1 bufferwould serve as well. The exact relations between ion concentrations, mobilities, pH's and pK's are given in reference ( 1 ) . Experimental Procedure (2)

Table 1). For analytical columns, glaw tubes 6 cm by 0.5 cm i d . , marked 2 cm from one end (t,he ton) are used. Place the tuhes in rubber

Have everything ready for overlayering (below). Prepare about 1 ml of lower gel solution per tube by mixing 1 part A to 1 p w t C to 2 parts fresh F (Table 1). The solutions should beat room temperaturelestbubhlesof air formwhile thegel

Table 1.

Solutions for the "Standard" Analytical Disc System

Lower Gel (A) $:sHCl TEMEDo water to 100 ml (BIT 8 . 9 ) (C) Acrvlamide Bisacrylamida water to '""- ~

.--. - ~Upper . . auner: -

~

2 4 . 0 ml 18.2 g 0 . 2 3 ml

'

30.0 g 0.8g

(B) 1 M HaPo. Tris TEMEDa water to 100 ml (pH 6 . 9 ) (Dl Acrylsmide Riracrybmide to 100 ml

... .

3 0 . 0 ~ 144.0g

1 N HCI = 83 ml cono. HCI/liter 1 M HaPo, = 6 8 . 3 ml 85% H~POdliter

Use 20 ml/liter final buffer (pH 8.3) Tracking dye: 0.001% Rromophenol blue in water Stainine solution: 1% Amido black in 7% hoetic acid

/

1 0 . 0 ~ 2.5.

-

Tris Glycine Water to 1000 ml

42

25.6 m l 5.7 g 0.46 ml

Journal o f Chemicd Education

Figure 2. An analytical disc electrophoresis apparatus. Conrtrvction is of Lucite. The numberr refer to: I l l upper electrode terminal; (2) tube rock; 10 X 3 X 9a/lsin.; the holes in the lower 9helf ore a/8 in. in diometer, those in the upper shelf ore "/a in. in diameter; 131 pouring spout in., projecting in. down; 14al locator for gar escape; 14) locator pins; pin holes; 151 lower electrode termind; the lower electrode is loosely coiled 18 gouge platinvm wire, soldered to b r a s terminals ct eoch end of the reservoir; metal other than platinum which is exposed to the lower b&r lower buffer should receive a thick coding of Lvcite glue or epoxy semsnt; 161 lower buffer reservoir; 3 X 3 X '/la in.; 171 gel tube; 181 No.3 rubber ~ t o ~ ~ 191 e r 20 ; X 150 mm bottomless test tube; 11 01 upper electrodes: 18 gouge plotinum wirer gealed into ' 1 4 in. a d . capillary tubing, leaving about I in. of bare platinum wire projecting down; the upper end of the plotinm is soldered to 2 in. wires which are attached to the bmsr bar with setscrews; NO. 3 rubber stoppers, bored to flt tightly over the capillary tubing, prein. brass bar. vent the electrode. from being immersed too deeply; I1 1 )

'Il6

'Ia

is polymerizing. Add the F last. Using a disposable pipet, add the gel solnt,ion t,ot,he2 em mark on the t,ubes,tapping t,hetuhea to insure that no air bubhles have been trapped in (,hem. Carefully over-layer the gel with 4-6 mm of water, using a drawn out Pastern pipet. The sharp boundary line will blur because of diffusion, but a new sharp line will appear ten mimkes t o one hour later, about 2 mm below t,he original interface. This indicaies that, thc gel has polymerized. Remove the upper liquid with a fliok of the wrist and place the s of tuhes near a fluorescent light. We use en a p p a r ~ t n consisting three stacked circular 30-watt, Cool Whit,e flnorescent lamps mounted on an aluminum plat,e with the hallast,~. This is convenient for polymerizat,ian of analytical and prepamhive gels (5) and can be constructed for about $50. Upper gel solution consists of 1 part B t,o 2 parts D t,o 1 part E to 4 parts water. Pipet 0.2 ml of this onto the t a p of each lower gel and then overlayer with 4-6 mm of solution E or water. Turn on the fluorescent lamp, and let the upper gel solntion photopolymerize for 30 minutes. It will be opalescent. Flick off t,he liquid. Do not leave the gels exposed to the lamp for excessive periods, ns heat from it t,end~t,o produce air bubbles in the gels, as well as convection in incomplet,ely polymerized regions. The gels mnst now be used wit,hin 3 hr or problems occur as a r e sult of diffusion bet,ween i.he upper and lower gels. If xtoppers have been used, insert, a. syringe needle between the stopper and tube t o prevent a vaeortm from forming when the stopper is pulled off. If t,here are air bubbles in the hot,tom of a gel tohe, remove them by adding buffer with al'asteurpipet. Fill the gel t,nhes up ribh npper buffer, dry the outsides of t,he t,uhes, and force t,hem through No. 3 rnbber stoppers which are bored to give a leak-proof fit. The at,oppers are forced on upside down. The stoppers are pushed into the bntt,om of 20 hy 150 mm glass t,est tnhes which have had t,he hoitoms removed, and a ponring spout made in the top. Add 700 ml of npper buffer t,o the lower buffer chamber. Set, t,he tube rack on the lower buffer chamher and place t,he gel tube assemblies in t,he rack so that. the gel tuhes poke through the holes and are in cont,act with t,he buffer. Add 1 ml of tracking dye to 200 ml of upper huffer. Fill the t,est tuhea with enough of t,his

solution t o make contact with the upper electrodes. Mske sure there are no air bubbles in the tops of the gel tubes. Conneot the electrodes with the power supply; positive polarity to the lower chamber electrode, negative t o the upper. In preparing samples for disc electrophoresis, i t is best to d i e lyze against the tris-glycine upper buffer. I n any event, the presence of large amounts of chloride, sulfate, etc., in the samples should be avoided, as they delay stacking for periods proportional t o the amount present. The dialyzed samples should be made 10% in sucrose, then layered onto the upper gels of appropriate tubes. A syringe with a 6in. needle is useful. About 50/#g of protein is applied per tube. Turn on the power supply and run a t 1.5/mA per tube for 1.5-3 hr, until t,he blue tracking dye has migrated to within 2-5 mrn of the bottom of the lower gel. Don't run the dye off the gel. Smaller i.d. tubes should he given less current or they

point.

Ificonstant-current power supply is used, the amperage

tste the t t b e as the needle moves farther into the tube. Rotate the tube when withdrawing the needle also. I t will probably he necessary t o rim the gel from both ends of the tube. Do not try to use a. longer needle as these are hard t o control. The needle should be attached t o a slow stream of water for lubrication during rimming. Alternatively, use an 18 gsuge needle insert with 8. rounded end; gel tubes are rimmed while immersed in water. If the eel is obstinate even after repeated rimming, use a rubber Pasteur\ipet bulb filled with water & force the gel&. Place the eel in the urotein stain solution for one hour. The

acid in hoth buffer chambers. Recently, Chrambach, et al. (4), have described a much simpler and more sensitive protein stain. The gels are soaked in 1% Coomassie blue blackin 12.5Y0 trichloroacetic acid. According to those authors, the uptake of stain by the zones of protein can be followed by eye. We have not tried this method. Ritchie, ct al. ( 6 ) , describe the causes and cures of some common anomalies in disc patterns. They avoid these by degassing their reagents, using a detergent (Tween 80) for better layering, and generally taking greater pains in preparing the gels. However, the above directions give satisfaotory reaults if followed carefully. Other Systems

While the standard system described above is most popular, disc electrophoresis can be set up with other ion systems (see Table 2). Further Information

In electrophoresis of nucleic acids, the sieving effect of the gels is the important factor in separations, since the charge to mass ratio of nucleic acids is constant, a t least a t neutral or alkaline pH. Staining for nucleic acids is done by soaking the gels overnight in 2% acridine orange, 1% lanthanum acetate, and 15y0 acetic acid (6). Removal of excess stain is by dialysis against 7% acetic acid. It is hard to quantitatively stain nucleic acids in gels, so instruments for measuring the ultraviolet absorption of nucleic acids in gels as a function of distance of migration have been developed. The const,ituents of acrylamide gels all absorb in t,he ultraviolet, so several techniques have been developed to reduce the "background": recrystallizing some of the reagents (7) (see below), electrophoresing the gels before applying any sample (7), and removing, then dialyzing the gels before re-inserting in the tubes and running the experiment (8).

Table 2. Some Other Disc Systems pH 4 . 3 : ? ! / a % gel: (stacks a t p l l 5.0)

(1) Runnine Stock solutions: as m standard, except for: (A) 1 N KOH 4 8 . 0 ml g + l _ s o e t i o acid 17.2 ml

(B) 48.0 ml

2.87 ml 0 . 4 6 ml water to 100 ml (pH 6 . 7 )

10X Concentrated upper buffer 8-alanine Glacial acetic acid H?O to 1 liter (,,A 5.0) Lorver gel: 1"art A to 2 parts C: to 1 part water to 4 parts F Upper gel:, 1 part I1 t o 2 parts D to 1 part E t o 4 parts water te: Run with electrodes reversed (HI

Running p!I: 2.3, 7f/1% pel: (stacks at p H 4.0) Stock aolutrons: a r m standard, e x e e ~ for: t (A) 1 N KOH 4 8 . 0 ml Glseisl acetic acid 2 1 3 . 0 ml TEMED 4.R ml Hz0 to 400 ml (pll 2 . Q ) (G) hmnronitsm permlfnte 1 . 4 go Il?O to 50 rnl (HI 1 0 X Conoentrated upper hafrer Glycine 28.1 9 Glacial aoetio acid 3.06 ml

(11) 48.0 rnl 2 . 9 5 ml 0.46 ml H10 t o 100 ml (pH 5.0)

The 71/2% standard gel is suit,able for proteins of molecular weight between 10,000 and 1,000,000 wit,h best resolution from 30,000 to 300,000 Daltons. For electrophoresis of substances of molecular weight less than 10,000 a smaller pore (higher acrylamide and methylene hisacrylamide ~oncent~ration) gel is used; up t,o 30%. For substances above 1,000,000 Daltons, a Z3/& gel is used. With 8 M urea-containing gels, t,he 7l/?9& st,andard concentration should he cut to SL/,O/, (3). If t,he acrylamide concentrat,ion is cut below S'%, t,he concent,rat,ionof met,hylene bisacrylamide should be increased, to 5% of the acrylamide concent,ration. The gels will then be opalescent. Some workers do not use an upper gel, hut simply layer their protein ont,o the lower gel. We believe use of the upper gel gives better result,s (see reference (5)). For people worried about cross-contaminat,ion between protein samples in different gel tubes, the protein may be mixed with a second sample of upper gel solut,ion,and t,he latt,er polymerized onto the st,acking gel. I n earlier literahre, t,hiswas called a "sample gel." However, we have had no difficulties with cross contaminat,ion using t,he method given in the directions. I n making up gels containing 8 M urea one can either add solid urea t,o the lower or upper gel solut.ions,or add concentrated stock solutions to 10 M u r e a . This is preferable to making all thc solutions 8 A[ in urea, since urea decomposes wit,h time to ammonium carbonate. The 10 M urea should he deionized by pouring t,hrough a short column of mixed-bed ion-exchange resin just before use (see Table 3). Ammonium persulfate is a strong oxidizing agent, and under some circumstances can produce inactivation of enzymes and even artifactual protein bands (9). This can be checked for in either of two ways: mix approximately 0.1 micromole of tris-thioglycolate with the protein before electrophoresis; or polymerize the lower gel with riboflavin and light. If the latter method is used, use the s:me concentration of riboflavin as is used in polymerizing the upper gels,; an excess inhibits polyVolume 46, Number 1, January 1969

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Table 3.

Solutions for 8 M Urea Gels

-

gel

rnl/ml gel solution

Imer: 5 X A 1.5XC 20 X F 10 M urea Upper:

5 X B 2 X I)" 2 X E' 10 M hrea 0.80 Pratein samnles should be made dense with urea rather than sucrose Solutions 1) and E may have to be heated (gently) before everything dissolves.

merization. Proteins electrophoresed in riboflavinpolymerized lower gels have higher R,'s than they have in persulfate polymerized ones. The time required for polymerization also varies wit,h the age of the solution F, so it should be made up fresh every week or so for reproducible polymerization rates. If recrystallized acrylamide is used, the amount of solution F used may have to be reduced. Since the polymerization is exothermic, it is better t,o avoid problems with convection by using minimal amounts of F consistent with polymerization in a reasonable time. The acrylamide polymerization occurs by a free radical mechanism. Consequently, the reaction goes faster a t higher temperatures, and is inhibited by a variety of "quenchers," including oxygen, sulfhydryl compounds, reducing agents in general, and some metal ions. Most commercial preparations of acrylamide contain enough impurities to retard polymerization, and some have enough to prevent it.. Acrylamide may be recrystallized by heating in benzene (ca. 100 g acrylamide per liter of benzene) to a temperature of 60-70°C. The impurities will lower the melting point of the undissolved acrylamide and will collect in the molten layer on the bottom of the beaker. Pour off the upper solution, and allow it to cool to room temperature. Do not chill, as benzene freezes a t 6°C. Collect the crystals of acrylamide by filtration. The impurity-rich material can be re-extracted with more benzene. Loening (7) recommends recrystallizing acrylamide from chloroform. In our experience the above method is more effective. Note that acrylamide vapors are quite poisonous and benzene vapor is not healthful either, SO DO THE RECRYSTALLIZATION I N A HOOD. Methylene bisacrylamide, TEMED, and ammonium persulfate are also poisonous, so avoid getting these reagents on external or internal body surfaces. Methylene bisacrylamide is recrystallized from acetone (7). Glycine can be recrystallized from hot water and tris from 95%,ethanol. The glass tubes are washed by soaking in chromic acid cleaning solution overnight, rinsed with glass-distilled water, soaked about a minute in 10% potassium hydroxide in 95% ethanol (w/v), and rinsed thoroughly, again in glass-distilled water. Dirty stoppers are suspended in glass-distilled water and potassium hydroxide pellets (5 g per 100 ml) added with stirring. After the solution cools to room temperature, the stoppers are rinsed in glass-distilled water, 0.1 N HCI, and twice more in glass-distilled water. 44

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Journal o f Chemical Education

ldentiflcation and Recovery

Often i t is useful to be able to determine whether a particular enzyme activity is associated with a certain protein band. One procedure is to slice the gel and try to elute the enzyme. Probably because the protein is enclosed within a three-dimensional lattice, this is rat.her difficult with acrylamide gels. Good results have been obtained by placing an acrylamide gel slice in a starch gel block and driving the protein into the starch by electrophoresis. Elution from the starch is much easier. For analysis of patterns obtained in electrophoresis of radioactive material, the gels are frozen in dry ice, sliced, the slices dissolved in hydrogen peroxide, and scintillation fluid added. If the gels are polymerized using ethylene diacrylate instead of methylene bisacrylamide, concentrated ammonium hydroxide can be used (10). There is a specific stain for dehydrogenases (Table 4). Table 4.

Dehvdroaenare Stain

Reagent

Amount

1 M tris, pH 9.0 10 mM DPNo 10 mg/ml N B P 0 . 4 rg/rnl PhlSo 10 mM substrate Water The reaeenls should be kent dark. a

IIPN, diphosphopyridine nucleotide NBT, nitro blue tetranolium. PMS, pbenasino methosulfate

The gels should be soaked in the dehydrogenase staining solution only 5-10 min for rings to appear a t the site of the dehydrogenases. Too long an incubation gives darker bands which blend into each other, making estimations of the number of bands difficult. Staining solutions have also been developed for leucine aminopeptidase and alkaline phosphatase (11), and RNA polymerase (18). Any colorimetric reaction for products can in theory be employed as a stain. More generally, soaking the gel in a solution containing a radioactively labeled competitive inhibitor, then dialyzing out the excess, should enable identification of any enzyme. This has been done successfully with a nuclease from corn roots which binds phosphate (J. M. Brewer and N. G. Sansing, unpublished data). Preparative Disc Electrophoresis

The constmction of the apparatus (See Fig. 3) and its operation are more extensively described in reference (3). It can be produced for less than $100 if a competent glass-blower is available. Several commercial apparatuses, based roughly on the same design, are available. Commercial power supplies of the wattage required run up to $1500. However, a constant-current power supply can be built for a third of that, and anonregulated one for $250. Cut a 7 cm diameter circle of 0.0016-in. dialysis tubing and immerse in water for a t least 5 min. Liberally coat with glycerol both the outside top edge of the bottom joint and inside bottom of the female ground glass surface on the bottom of the lower buffer reservoir. Center the dialysis tubing on the bottom joint, and

Figure 3 . A preparative disc electrophoresis opparatur. The numbers refer to: I l l lower buffer bath 113 I); 121 capillary tvbe section of upper resenoir os%embly; 131 vpper buffer rsrenoir of upper reservoir assembly; (4) c o o l m t inlet, and outletr; 151 lower buffer reservoir; ( 6 ) bottom ioint; 171 plastic tripod; 18) dialysis membrane; ( 9 ) g e l ; 110) areas where coolant circulates; 11 11 elution buffer inlet; 11 2) O-rings; (1 3 ) capillmy tvbe Ito pump and fraction collector).

without,twisting, slowly force the lower buffer reservoir onto it. The membrane should be unwrinkled. Push this apparatus down into the lower buffer bath, and check for leaks of air through or around the membrane. If any are seen, take the apparatus out of the bath, and force the lower buffer reservoir more tightly onto the bottom joint. If this does not eliminate leakage, take the sections apart, discard the membrane, and try again with a fresh circle. When a leak-free apparatus has been assembled, use a d-shaped piece of glass tubing to withdraw all air from beneath t,he membrane, and sink the apparatus in the bath so that it sits on the plastic tripod. Insert the capillary tube section into the upper reservoir. Seat the O-ring so that the lower ends are the same length. This is the upper reservoir assembly. Place parafilm over the bottom of the assembly and temporarily secure with a rubber band. Any parafilm extending more than an inch up the sides should be cut off with a razor blade. Attach the evened edge of the parafilm to the glass with electrical tape (the black plastic variety is best). Make sure the capillary centers on the parafilm. Clamp the assembly upright, with the bottom resting on a Petri dish, and pour in the lower gel solution (Table 5). Layer on 10 ml of water. A syringe with the needle attached to a long plastic tube, 0.5 mm i.d., is convenient. After 1 hr in the cold room (or less at room tempera-

ture), the lower gel sets and becomes slightly opaque with a sharp boundary line. Remove the upper liquid with the syringe, rinse the lower gel with water, then with two successive 4.5 ml portions of upper gel solution. Add the remaining 15 ml of upper gel solution, layer on 10 ml of water and photopolymerize with a circular fluorescent lamp. (The lamp will not light if it is cold, so if polymerizing in the cold room, it must be put in the cold room immediately before use.) After the upper gel has polymerized (0.5-1 hr for firmness), pour off the upper liquid, rinse with upper buffer, and carefully remove the tape and parafilm. Check to see that the bottom of the capillary tube section and the capillary tube itself is free of gel. If they aren't, carefully remove such gel: a small piece of gel can plug the capillary tube and ruin the experiment. As the upper reservoir assembly is lowered into the lower reservoir, add upper buffer on top of the gel to maintain hydrostatic equilibrium. When the O-ring seats in the lower reservoir, adjust the gel-membrane distance to less than 1 mm. Then connect the tube from the elution (lower buffer) bottle, and connect the coolant tubing. The entire apparatus is now sitting on the plastic tripod. Make sure that the apparatus is vertical, add one ml of tracking dye, and layer on the protein-10% sucrose solution with the syringe. Usually, 10-50 mg of protein is applied, but if the protein is very heterogeneous up to 100 mg may he electrophoresed. Connect the electrodes and run a t 20 rnA (or less, if mixing in the protein solution is observed) until the tracking dye enters the upper gel. Then set the amperage to 30-50 mA and collect fractions. Keep the flow rate greater than 0.2 ml/min. After the run, disconnect the electrodes and coolant, remove the upper reservoir and discard the gel and upper buffer. The lower reservoir can be submerged and used again, as long as the membrane does not leak. Preparative-scale disc electrophoresis should be accompanied by control experiments on an analytical scale, both beforehand and on the individual proteins isolated using the preparative apparatus. Acknowledgment

Partial support for this paper was provided by a National Science Foundation Grant (GB 5918) to J.M.B. We arc indebted to Dr. R. Prairie, whose instructions for disc clcct~rophoresisare the origi~rsof ours. Literature Cited ( 1 ) O R N S T ~ NL, . , 1V. Y. Acad. Annals, 121, 321 (1964). (2) I).~vrs,B . J., N.Y. Aead. Annals, 121,404 (1964). (3) JOYIN, T., CHRAMBACH, A,, AND NAUOATON,M. A,, Anal. Biochem., 9,351 (1904). (4) CHRAMIII.CH, A,, R,EISFELD,R. A., WYCKDFF,M., >\NU Znccnm., J..Anal. ~ , Hiorhem..,20.. 150 (19671. , , (5) RITCHII:,R. F., HARTIIR,G., A N D BAYLES, T. B., J . Lab. Clin. Med., 68,842 (1966). (6) R l c r r ~ n ~ E. s , C., COLL,J. A,, AND GBATZKR, M. A,, A n d . Biochem., 12,452 (1965). (7) LOENIND, (1. E., Bioehem. J., 102, 251 (1967). (8) BTSHOP, 1). H. L . , ~ L A Y I I R O O KJ., R., AND ~ P I K G E L M - 4 Ns ,. , J . Mol. Hid.. 26. 373 (1967). (9) B~x\\-xa, J. M.; S&xce,'l56,256 (1967). , WILLEMS, M., AND PKN(10) WJ:INH!~RD,R . A,, ~ E N I N D U., MAN, S., Pn'AS, 58, 1088 (1967). (11) LAW,G. R . J., Science, 156,1106 (1967). (12) K L I ~C., B . , JBC, 242,3579 (1967). ~~

Table 5.

Solutions for Preparative Disc Electrophoresis

Upper bnffer: as in the standard snnlytiral system Lower and eInt,iun buffer: 12 N HCI 65 ml Tris (TIIAM) 160 g Water to 13 1 Lower gel Up er gel k i ~ u t i o nB Solotian A 12 5 r n ~ Sololion 1) Solution C 12.5 Solution E Solution P (70 mg) 25 0

3 . 0 m~ 6.0 3 .0

Volume 46, Number 1, January 1969

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