A simple preparative polyacrylamide disc gel electrophoresis apparatus: Purification of three branched-chain amino acid binding proteins from Escherichia coli

ANALYTICAL

5, 297-311

BIOCHEMISTRY

A Simple

(1973)

Preparative

Electrophoresis Three

Polyacrylamide Apparatus:

Branched-Chain

Disc Gel

Purification Amino

of

Acid

Binding Proteins from Escherichia colil CLEMENT RANDALL Department

of

E. FURLONG, CERVIN CIRAKOGLU,z C. WILLIS, AND PATRICIA A. SANTY3

Biochemistry, Received

University May

of

25, 1972;

California,

accepted

Riverside, August

California

92602

9, 1972

The equipment used for preparative polyacrylamide gel electrophoresis has been either difficult to construct or costly if purchased commercially. An inexpensive preparative acrylamide gel apparatus and peristaltic pump are described in this paper which are easy to use and may be constructed from readily available materials. The construction of the preparative gel apparatus requires no special machining or glass blowing. This report describes the use of the disc gel apparatus in the final purification step of three binding proteins which appear to be involved in the transport of the branched-chain amino acids in Escherichia di. Two of these proteins have been described ‘previously (l-4). The apparatus has also been successfully used in a number of other laboratories for the purification of a variety of other proteins (5-9). METHODS

Reagents Necessary for Preparative Gel Electrophoresis. The reagents necessary to operate the apparatus are the components of the chosen buffer system. For the modified Omstein-Davis system (llJ2) described here, the following chemicals are used: Tris buffer, phosphoric acid, N,N,N’,N’-tetramethylethylenediamine (TEMED) , hydrochloric acid, flavin mononucleotide, glycine, ammonium persulfate, thioglycolic acid, ‘This work was supported in part by PHS Research National Institute of General Medical Sciences. ‘Predoctoral fellow of the Agency for International ‘Recipient of a Summer Research Fellowship from 297 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

grant

No.

the

Development. Calbiochem.

GM

13311

from

298

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ET

AL.

sucrose or glycerol, acrylamide and bisacrylamide (N,N’-methylenebisacrylamide). For other buffer systems see Refs. 10 and 14. Suggested Equipment. In addition to the materials listed above, the following items are required or recommended: a fraction collector, a power supply (lo-30 mA at 2OCL300 V), and source of coolant. A uv monitoring device is also useful as it is with most fractionation equipment; however, it is certainly not a necessity. A peristalic pump such as the simple one described below is also useful although the flow of elution buffer may be regulated with a screw clamp. Construction of the Preparative Gel Apparatus. The materials necessary for the construction of the apparatus are listed in Table 1. The assembled

i\interials

TABLE to Construct Part (Fig. I)

Material

1 Apparatus

Number of pieces

Dimensions Length (in.)

Tygon or other tubing”

vinyl

plastic

Pyrex glass tubing (10) Glass or Plexiglas t,ubing Polypropylene tee Platinum wire Plexiglas sheet Rubber stopper Polyethylene beakers (or suit able reservoirs) Stainless steel needle Elution reservoir Banana plugs Dialysis tubing Cyclohexanone Chloroform or plastic glue Polyethylene tubing

a

Inner

Outer

I

6

1%

l$$-l~~

1 2

2’i

1

1’4

2

l-6 8

% 0 025b 24 mm

1%

74

h 1

1 1 1 2 1 1

j, k

2

m n

1 1

0

2

P

-

Screw clamp Razor blade Syringe Q The larger sizes are available from most * Available from Technicon Instruments,

Diameter (in.)

2

26 mm

1 ?8

26 gauge,

6 in.

‘4 x llq x 1” /4 in. f7 600 ml vol (or larger for runs) 22 gauge e.g., 1 liter polyethylene male or female at least lJ$ in. wide approx 5 ml approx 1 ml 1 ft, 3s in. diam 1 ft, 34 in. diam

3-10 vendors Tarrytown,

56

longer

bottle

ml

of commercial NY, part

plastics. number

116-0533-06.

PREPARATIVE

GEL

299

APPARATUS

apparatus with component parts labeled a-p is shown in Fig. 1. The ends of the large vinyl chloride tube (6 X l-1/4 X 1-3h in.) are squarely trimmed using a band saw or a razor blade dipped in glycerol to form part a. A piece of z/,-in. paper tape wrapped around the tubing serves as a cutting guide. Crooked tubing may be straightened before trimming by briefly autoclaving or heating in boiling water. Holes are cut on opposing sides of the tube, l/z in. from either end using a cork borer. The holes should be slight’ly smaller than the diameter of the tubing (3/s in.) which is next glued into the holes, using cyclohexanone (parts c). The glued joints are allowed to set before attaching part b. Part b is formed from the 21/ X 1 X 11/4-in. tygon tube by wrapping each end with s/,-in. tape, leaving I/ in. exposed, and cutting both ends of the tube at a 45” angle with a glycerol-lubricated razor blade. Insertion of a glycerol-lubricated test tube to hold the tubing rigid will facilitate the cutting. Eit’her of the small beveled pieces of tubing resulting from this cutting will serve as the water-jacket seal (part d). The outside of

and

FIG. 1. Diagram of the preparative other information are included

polyacrylamide in Table 1.

gel

apparatus.

The

dimensions

300

FURLONG

ET

AL.

one end of the l-s/4-in. tube (part b) and the inside of one end of the 6-in. tube (part a) are each wetted with cyclohexanone about 3/ in. from the end. The wetted end of the smaller tube is then inserted about 3/8 in. into the wetted end of the larger tube. Any pressure required to seal the two pieces is applied by simply squeezing the two surfaces together. It is easy to see where good contact is being made and where more cyclohexanone is required. Additional cyclohexanone may be added if needed using the tip of a spatula. For some applications the cooling jacket may be omitted; particularly with very small-diameter columns ut,ilizing the same type of elution chamber. After the glued joint between parts a and b has set for l-2 hr, the holes are cut for the elution tubes (parts e). A small-diameter, sharpened metal tube (e.g., 13 gauge syringe needle) will serve to cut the holes which should be slightly smaller than the outside diameter of the small tubes. The relative position of these holes through b is shown in Fig. 1. They should be 180” apart and as close to the edge of tube a as possible. The elution tubes are then glued in these holes using cyclohexanone. It is best to let them protrude a little into the cavity of tube b and to cut them exactly flush with the inside of the tube after the glue has set. A scalpel is useful for this trimming operation. After the ends of the elution tubes are trimmed, ys-in. pieces of a 22 gauge stainless steel needle (cut with a file) are inserted with forceps into the elution tubes from the inside cavity of tube b forming parts m. About $& in. is left protruding into the cavity of tube b to prevent the occlusion of the elution tubes by the column (f) or the gel-buffer bridge (g). Glass tubing (1 in. long, 25-26 mm o.d.) may be used for the salt bridge; or a Plexiglas bridge may be constructed by drilling a s/s-in. hole in the center of the l-1/4 X l-1/ X l/s-in. Plexiglas sheet and trimming the edge of the sheet to the outside diameter of the Plexiglas tube. The disc is then attached to a chloroform-wetted end of the Plexiglas tube. The resulting shoulder (part h) prevents the gel from sliding out the bottom of the buffer bridge. The water-layering tube (part i) is made by heating and drawing out a piece of I$&in. polyethylene tubing in much the same way that glass tubing is drawn out. The smaller diameter of the drawn-out tubing will allow a slow flow rate to prevent mixing the gel. Small-diameter tubing may be prepared from the l/4 in. diameter polyethylene tubing in the same way. This tubing may be used to connect the elution reservoir to the tee and the elution tube through the pump tube to the fraction collector. Some practice is necessary in order to produce uniform tubing. Polyethylene beakers or other suitable containers are used to construct the upper and lower buffer reservoirs. A hole of appropriate diameter is

PREPARATIVE

GEL

APPARATUS

301

drilled 1/4 in. below the top of the beaker. The male or female banana plug connector is inserted through the hole and a nut is used to secure the plug to the beaker. A second nut is used to attach a piece of 26 gauge platinum wire, which extends to the bottom of the beaker (part j). The electrodes may also be positioned in the center of the reservoirs. The upper reservoir (part k) is made in the same manner, except that a l-s/ in. diameter hole is cut in the bottom center of the beaker to accommodate the gel column. The sharp outer edges of the ends of the 8 in. long Pyrex glass tube (part f) are gently fire polished. If this is not done, the column will cut the plastic tapered shoulder of tube b during assembly of the apparatus. It is important that the fit of the glass tubing (parts f and g) into the Tygon tubing (part b) is tested before beginning construction to assure proper sealing, since there is occasional variation in tubing sizes. Operation of the Preparative Gel Apparatus with a Discontinuous Buffer System. One end of the well-cleaned long Pyrex (9) glass tube (part f) is covered with Parafilm. The Parafilm may be sealed to. the end of the tube with either an O-ring or by wrapping the Parafilm tightly upon itself. In either case, the Parafilm covering the end must be free of folds or wrinkles. It is extremely important to have the tube very clean; otherwise, the gel may release from the glass walls during operation. The covered end is placed on a flat, leveled glass or rubber surface in a 4°C room or under ice water (the gel should be polymerized at the running temperature) and cold deaerated resolving gel-buffer solution of the desired percentage gel is pipetted into the tube of a height of from 1 to 6 cm immediately after mixing and deaeration. (See Refs. 10 and 13 for a discussion of the effect gel concentration on protein mobility. Longer gels take a very long time to run. A gel should, therefore, be just long enough to achieve the desired resolution.) The waterlayering device (part i) is immediately used to gently cover the resolving gel solution with a layer of water in order to provide a flat gel surface. The device described is easy to use and can provide a very slow flow rate to prevent mixing of the water with the gel. The flow rate is determined by adjusting the height of tube i. After the gel has polymerized, the water layer is removed. A syringe fitted with a long needle or tubing is useful here. Care should be taken not to nick the gel. The top of the resolving gel is gently rinsed twice with l/2-ml portions of the stacking gel solution. A volume of stacking gel soIution slightly in excess of the volume of the sample to the applied (approx 20% larger volume) is introduced on top of the resolving gel and layered with water as before. The gel is photopolymerized using a fluorescent lamp. After polymerization, the water is again removed. Extreme care must be taken in this

302

FURLONG

ET

AL.

step, since the stacking gel is quite fragile. The top of the stacking gel is rinsed twice with $&ml portions of upper buffer. The Parafilm covering the end of column f is now carefully removed, and the gel column is inserted through the cooling jacket into the wetted elution collar (Fig. 1) as far down as the stainless steel inserts. The beveled top of the elution chamber accepts the gel coumn easily. The wetted water jacket seal (part d) is now inserted between the top of the cooling jacket and the gel column. The buffer-gel plug is prepared at the same time that the resolving gel is polymerized by covering the bottom end of the short glass or Plexiglas tube (parts g and h) with Parafilm. A l-cm piece of 1 X 1/,-in. plastic tubing is slipped over the upper end of this tube to serve as an extender. The tube is then filled nearly to the top of extension with an approximately 87% gel solution and layered with water. After the gel has polymerized, the extender is removed and a water-wetted razor blade is used to squarely cut the gel even with the top of the tube. Several drops of elution buffer are placed on top of the gel to prevent bubble formation and a 1-1,~ in. diameter disc of dialysis tubing is centered over the top of the gel plug. The elution chamber is very slowly pushed down over the gel plug tube until the membrane just touches the stainless steel elution tube inserts (Fig. 1). Care should be taken not to dislodge the gel during this operation. Filling the bridge with acrylamide prevents the net flow of buffer across the dialysis membrane. Drawn-out polyethylene tubing or other small-diameter tubing is connected to a reservoir (part n) containing deaerated elution buffer and inserted into the small tee connected to one of the two elution tubes. A piece of tubing extends from the tee to the top of the upper reservoir and is held in place with a rubber band around the upper reservior. This tube serves as a measure of the hydrostatic pressure pushing up on the gel as well as a bubble trap. Another piece of small-diameter tubing is inserted into the other elution tube and leads through the pump to the fraction collector. Deaerated elution buffer is allowed to flow slowly through the elution tube into the elution chamber below the resolving gel and out the tube leading to the fraction collector until all of the bubbles are removed from the narrow elution chamber. It will be necessary to carefully tilt the column to eliminate bubbles from the elution chamber. A clamp or the pump is used to stop or regulate the buffer flow. Care must be taken to prevent upward or downward pressure on the gel which would cause t’he release of the gel from the glass column. The bored out rubber stopper (part 1) is placed over the upper end >f the gel column followed by the upper reservoir (part k). The lower buffer reservoir is filled with lower buffer, and the completely assembled column

PREPARATIVE

GEL

APPARATUS

303

is firmly clamped in place above it with buffer bridge extending down into the lower buffer. The buffer level should not reach the banana plug connectors. The top of the cooling jacket is a convenient place to attach a two-jawed clamp. The tube leading from the tee held against the upper reservoir with a rubber band allows the adjustment of the level of the elution buffer to compensate for the downward pressure of the upper buffer. The placement of the elution buffer reservior (part n) should be such that the level of buffer in the tube attached to the upper resevoir should be slightly higher (approx 1 cm) than the upper buffer. A Mariotte bottle may be used to avoid changing the level of the reservoir during the run. The cooling water is connected through the cooling jacket and care is taken that the column is level. The gel column and the upper reservoir are now filled with upper buffer.4 The water-layering tube may be used to begin with so that the stacking gel is not damaged. The elution reservoir should be raised slowly to prevent a differential hydrostatic pressure on the gel. It is a good policy to check the flow of t’he elution buffer through the elution chamber prior to the introduction of the sample. The flow of the elution buffer may be regulated by a screw clamp and gravity flow or a peristaltic pump may be inserted between the apparatus and the fraction collector. A simple variable speed peristaltic pump is described below. Buffer System. There are many different buffer solutions used in preparative polyacrylamide gel electrophoresis. Chrambach and Rodbard have written an excellent review covering most aspects of gel electrophoresis (10). In addition to the above reference, the bibliography of abstracts compiled by B. J. Haywood is an excellent source for different buffer systems (14). Shuster has written a general discussion of preparative gel electrophoresis (15). The discontinuous system described below is a modification of the Ornstein-Davis (11,12) system and has been found to work with a great number of different proteins. We have also used a Tris-Tricine system which incorporates lower pH values (3). To form the resolving gel, we have found it convenient to mix one part gel (four times the desired final acrylamide concentration), one part buffer, and two parts catalyst. For example, to prepare a 6% gel resolving gel with 0.375 M Tris-HCI buffer (pH 8.9)) one part 32% gel (1: 1.5 dilution of a 48% acrylamide-1.6% bis acrylamide stock solution) is mixed ‘We have also used a buffer replenishment method for long runs which is achieved simply by pumping fresh buffer slowly into the reservoir and letting excess buffer exit through an overflow tube inserted in the sides of the reservoirs at the level of the buffers. A glass rod down the center of the column also helps to stabilize the gel during a long run.

304

FURLONG

ET

AL.

with one part of 1.5 M Tris-HCl (pH 8.9) :0.3% v/v TEMED and two parts 0.25% ammonium persulfate. The gel solution for the salt bridge is formed by adding one part of the stock gel solution (ate least 32% acrylamide monomer-l% bisacrylamide) to one part 0.4 M Tris-HCl (pH 8.1) :0.3% v/v TEMED and two parts 0.25% ammonium persulfate. The stacking gel solution is similarly prepared by mixing two parts 5% acrylamide-1.25% bisacrylamidc with one part 0.238 M Tris-0.128 M H,PO, (pH 7.2) :O.l% v/v TEMED, and one part catalyst solution which cont,ains (per 100 ml) 3 mg flavin mononucleotide and 40 g sucrose (this solution should be filter sterilized and stored at 4°C in the dark or stored frozen). The upper buffer is 0.052 M Tris-HCl, 0.055 M glycine (pH 8.9)) 1 mM thioglycolate and the lower and elut,ion buffers are O.lOM Tris-HCl (pH 8.1). The upper buffer and the 1:4 dilution of stock stacking gel buffer, against which samples are dialyzed, routinely contain 1 mM thioglycolate or reduced glutathione (16). It is necessary to have the sulfhydryl protector charged so that it will migrate through the gel. When necessary, the elution buffer contains I mM betamercaptoethanol. The samples are of the same buffer composition as the stacking gel. This is achieved by adding a concentrated solution of stacking gel buffer to the sample; by dialysis of the sample against an appropriate dilution of stacking gel buffer; by buffer exchange during concentration of sample in a commercial protein concentration apparatus such as an Amicon ultrafiltration apparatus; or by buffer exchange chromatography followed by ultrafiltrat,ion. To insure adequate drnsity of the sample either sucrose or glycerol is added to 10% final concentration. The addition of 0.001-0.050 ml of a 0.01% solution of bromphenol blue to the sample will provide a dye band marker for a visual check on the progress of the run The amount. of dye may be adjusted to keep the recorder pen on scale if the runs are continuously monitored. The sample is slowly introduced under the upper buffer just above the stacking gel with the use of a syringe fitted with a long piece of polyethylene tubing or wit’h a peristaltic pump. Care is again taken not’ to injure the stacking gel or to mix the protein solution with the upper buffer. The electrodes are connected to the power supply with the anode at the bottom and current, is applied to the apparatus.” The allowable current is determined by observing the top of the sample. If it becomes wavy, too much current is being applied. The stacking voltage is usually about 200-300V at ‘The reasons

buffer reservoirs of safety.

arc

usually

covered

during

the

run

with

plastic

lids

for

PREPARATIVE

GEL

APPARATUS

305

about 15-20 mA. The running voltage may be higher. The collection of fractions should be started just before the dye marker comes off the column. The fraction size is usually 2-3 ml; however, this varies depending upon the particular application. The buffer flow rate is usually 0.25-1 ml per minute. The apparatus may also be used with a continuous buffer system. The use of the apparatus with detergent or low-percentage gel may require the bottom of the gel to be supported. In these cases, it is best not to glue tube a to b. Instead, the upper bevel should be out in the opposite direction and a disc of 400 nylon mesh inserted,over the lower end of the gel column in the same manner as the dialysis membrane is placed over the buffer bridge. This will prevent the gel from sliding into the narrow elution chamber. The bevel allows the cooling jacket to be slipped over the gel column after the column is inserted into the elution collar. If the outside diameter of tube b is not large enough to seal well against tube a, some type of circular band clamp should be used to tighten them together. Peristaltic Pump. Figure 2 shows a simple, variable-speed peristaltic pump constructed from a dc gear reduction motor. The motor is mounted to a l/-in. Plexiglas or metal base such that the motor may be easily moved backward or forward to apply more or less pressure on the pump tube(s). Two Flexaframe feet, (Fisher) are attached to a short l/z in. diameter aluminum rod which is drilled coaxially to accept the motor shaft. A hole is drilled perpendicularly in the rod so that the set screw from the foot (c) nearest the motor tightens directly aga@t the motor shaft. The set screw on the foot away from the motor (d) tightens into a shallow hole in the shaft (e) to prevent the feet, from slipping relative to each other. The rollers (3/1s in. diam steel or st$@less steel rods) are passed through the enlarged existing screw holes in .the Flexaframe feet and washers are soldered to the ends of the rods (g). It should also be possible to insert small ball bearings in the screw holes by machining them with a flat end mill. However, we have found this quickly constructed pump to function very well without bearings provided that the roller ends are oiled occasionally. The manifold tubing available from Technicon, Tarrytown, NY, part number 116-0533-19 (i.d., 0.060 in.), works well;.‘Gith this particular pump. The tubing has ‘Lstops” already attached to p’revent slippage through slit a. The arrangement of the tubing is shown in Fig. 2. Several tubes of the same or larger diameters may be pumped at the same time. The distance between e and a is adjusted so that the tubing must be stretched tightly around the pump head. A brace is used to prevent the tubing support from bending. A beveled slit of I/is in. at the front (a)

306

FIG.

struction

FURLONG

2A

(top), B (bottom). is discussed in the

Photographs text.

ET

AL.

of the

peristaltic

pump.

The

pump

PREPARATIVE

GEL

307

APPARATUS

and s/s in. (b) at the back has been found to be sufficient for several tubing sizes. Note that the slits are tapered so that the tubing is not closed off by pulling across a sharp edge. The speed of the pump is regulated either by a variable transformer or a solid-state power control. A small plastic fan on the main motor shaft cools the motor. RESULTS

Purification

of the Branched Chain Amino Acid Binding Proteins

The column (2.4 cm i.d.) was prepared as described. A 5 cm high, 7.5% resolving gel was used with a 0.5 cm high stacking gel (volume of stacking gel N 1.2 times volume of sample). The sample was equilibrated with stacking gel buffer in an Amicon ultrafiltration apparatus using a UM-10 membrane. A sample size of 0.8 ml containing 30.4 mg protein from the pooled leucine-isoleucine-valine binding (LIV-BP) fractions of DEAEcellulose-chromatographed shock fluid from Escherichia coli W3092 (3,4) was layered under the upper buffer. The sample also contained 2 ~1 of a 0.01% solution of bromphenol blue, 8 ~1 of 100 mM thioglycolate, and 30 ~1 of 50% glycerol. Electrophoresis was carried out with the anode at the bottom reservoir at a constant voltage of 18OV with a starting amperage of 17.5 mA

0

IO

20

30

40

Fractton

50

60

70

60

90

Number

FIG. 3. El&ion profile of the preparative polyacrylamide electrophoresis of the pooled DEAE fractions containing primarily LIV-BP. The broken line indicates the absorbance at 280 nm. The solid lines indicate the binding activities by membrane filter assays (3). The open circles represent leucine binding, the squares isoleucine binding and the triangles valine binding.

308

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AL.

provided by a Heathkit high-voltage power supply. Simple power supplies similar to the one described by Davis and Ornstein have also been used. (Current regulated power supplies are more desirable ; however, they are also much more expensive.) The flow rate of the elution buffer was 0.7 ml per minute. Fractions of 2.5 ml were collected. Absorbance was continuously monitored at 280 nm. The elution profile is shown in Fig. 3. Collection of fractions was begun just before the bromphenol blue reached the bottom of the gel column. The first branched-chain amino acid binding protein to emerge was the leucine-specific binding protein (LS-BP) (3,4). The second binding protein to emerge was a new member of the family of branched-chain amino acid binding proteins which preferentially binds isoleucine. The characterization of this protein will be described elsewhere. The last protein to emerge was the leucine-isoleucine-valine binding protein (LIV-BP) U-4) *

FIG. 4. Analytical disc gels of selected fractions from the elution profile shown in Fig. 3. Buffers and gel concentrations are identical to those used in preparative electrophoresis and are described in the text. The analytical gels (5 mm i.d.) contained a 5-cm resolving gel and a l-cm stacking gel. Sample volumes were 200 ~1. Electrophoresis was performed at 22°C with a current of 4 mA per tube for 60 min. Gels were stained with Coomassie blue (17). S.M. is the pooled DEAEcellulose fractions. The numbers under the gels refer to the fractions analyzed. Samples of SM., fractions 10, 12, 15, and 22 contained approximately 80 pg of protein while samples of fractions 42, 51, 63, and 76 contained approximately 20 pg of protein.

PREPARATIVE

GEL

APPARATUS

309

Analytical disc gels were run on various fractions which eluted from the gel apparatus. The results are shown in Fig. 4. The slowest moving component appeared to be the periplasmic asparaginase (16). This protein is most conveniently purified with the use of shorter gels. The overall yield of binding activity was greater than 50%. The yields with a number of proteins varied between 50 and 100% (l-8). This particular run resulted in a six-fold purification of the (LS-BP) and an eight-fold purification of the LIV-BP. Analytical gels of the pure branched chain binding proteins are shown in Fig. 5. The proteins shown in Fig. 5 were purified by preparative disk gel electrophoresis of more highly purified starting material (3,4). DISCUSSION

Preparative polyacrylamide gel electrophoresis has proven to be a powerful fractionation procedure for macromolecules (10,15). The high cost of commerically available equipment has led us to develop the simple, inexpensive instrument described here. This apparatus is easy to

FIG. 5. Analytical disc gels of the designated proteins: L, leucine specific binding protein (3), LIV, leucine-isoleucine-valine binding protein (l-4), I, Isoleucine binding protein; LIV-I-L, mixture of the three proteins. The purified isoleucine binding protein contains a trace of the jTJJ’-BP.

310

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AL.

construct and operate, and may be used to prepare milligram quantities of protein. It is an ideal device for learning preparative disc gel electrophoresis as well as establishing optimal conditions for use of a larger apparatus. Smaller apparatus may be constructed using the same design shown here. Larger instruments of this design should incorporate central cooling and radial elution. We have found this medium size apparatus to be useful for the purification of a number of bacterial binding proteins (3,4). Anderson and McCarty used an earlier version of this apparatus in the purification of plastocyanin from spinach chloroplasts. Ribereau and Preiss used this apparatus in determining the optimal conditions (6) for large-scale purification of ADP-glucose pyrophosphorylase from spinach leaves (7). Witheiler and Wilson used it for the purification of a peptidase from sheep red cells (8) and Granger and Kolb used it for the final step in the purification of lymphotoxin secreted by human lymphocytes (9). The construction and use electrophoresis apparatus and apparatus in the purification proteins from Eschetichia coli

of a simple preparative polyacrylamide gel peristaltic pump is described. The use of the of three branched-chain amino acid binding is demonstrated.

ACKNOWLEDGMENTS The authors are grateful to Mr. William Speck for photographical assistance and to Dr. L. M. Shannon and Mr. Richard G. Morris for their helpful suggestions in the preparation of this manuscript. REFERENCES 1. PENROSE, J. Biol. 2. ANRAKU, 3. FURLONG,

W. R., NICHOALDS, G. E., PIPERNO, J. R., AND OXENDER, Chem. 243, 5921. Y. (1968) J. Biol. Chem. 243, 3116.

D.

L. (1968)

C. E., AND WEINER, J. H. (1970) Biochem. Biophys. Res. Commun. 38, 1076. 4. FURMNG, C. E., AND HEPPEL, L. A. (1971) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), XVIIB, p. 639, Academic Press, New York. 5. ANDERSON, M. M., AND MCCARTY, R. E. (1969) Biochim. Biophys. Acta 189, 193. 6. RIB~REAU-GAYON, G., AND PREISS, J., personal communication. 7. RIF&REAU-GAYON, G., AND PREISS, J. (1971) in Methods in Enzymology (Colowick, S. P., and Kaplan. N. O., ed.), Vol. XXIII, p. 618, Academic Press, New York. 8. WITHEILER, J., AND WILSON, D. B. (1972) J. Biol. Chem. 247, 2217. 9. GRANGER, G. A., AND KOLB, W. P., personal communication. 19. CHRAMBACH, A., AND RODBARD, D. (1971) Science 172, 440. 11. ORNSTEIN, L. (1964) Ann. N. Y. Acad. Sci. 121, 321. 12. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404. ’ 13. HEDRICH, J. L., AND SMITH, A. J. (1968) Arch. Biochem. Biophys. 126, 155.

PREPARATIVE

GEL

APPARATUS

311

14. HAYWARD, B. J. (1969) Electrophoresis Technical Applications-a Bibliography of Abstracts, Ann Arbor-Humphrey Science Publishers, Ann Arbor, MI. 15. SCHUSTER, L. (1971) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. XXII, p. 412, Academic Press, New York. 16. BREWER, J. M. (1967) Science 156, 256. 17. WILLIS, R. C., unpublished results. 18. CHRAMBACH, A., REISFELD, R. A., WYCICOFF, M., AND ZACCARI, J. (1967) Anal. Biochem. 20, 150.