ANALYTICAL BIOCHEMISTRY 99, 233-241 (1979)
A Miniaturized System for Electrophoresis on Polyacrylamide Gels t,2 Z E N - I C H I OGITA* AND C L E M E N T L . MARKERT'~
*Research Institute for Oriental Medicines, University of Toyama, Gofuku, Toyama, 930 Japan, and tDepartment of Biology, Yale Unif~ersity, New Haven, Connecticut 06520 Received May 29, 1979 The design, construction, and use of a simple economical system for gel electrophoresis are described, along with effective procedures and the required materials. Examples of successful use of the system are presented.
Polyacrylamide gel electrophoresis, since its introduction in 1959 by Raymond and Weintraub (1), has proved to be a useful and powerful method for separating proteins and nucleic acids. The system has been extended for the estimation of molecular weights of polypeptide chains by using the anionic detergent, sodium dodecyl sulfate (SDS) 3 (2-4). Recently O'Farrell (5) and Scheele (6) have described a two-dimensional polyacrylamide gel electrophoretic technique that effectively resolves complex protein mixtures obtained from tissue homogenates. However, we have not found these methods as published to be satisfactory for resolving and measuring enzymes at very low concentrations. Therefore, we developed an improved miniaturized system with greater sensitivity and high resolving power. 1 This work was supported in part by a Scientific Research Grant of the Ministry of Education of Japan, by a Grant of the Ministry of Health and Welfare of Japan, and by NIH Biomedical Research Support grant 5-S07-RR-07015. 2 This paper is dedicated to the memory of Dr. Alvin Nason. 3 Abbreviations used: SDS, sodium dodecyl sulfate; Bis, N,N'-methylenebisacrylamide; TEMED, N, N,N',N'-tetramethylenediamine; TCA, trichloroacetic acid; LDH, lactate dehydrogenase; NBT, nitroblue tetrazo|ium; PMS, phenazine methosulfate; tlc, thin-layer chromatography; BPB, bromophenol blue.
In this paper we describe this miniaturized polyacrylamide vertical slab gel electrophoretic system. It is simple to construct and economical, resolves 3-/A samples of protein solutions, and is a considerable improvement on many polyacrylamide systems now in common use. We have tested the system in the analysis of the esterases in homogenates of mouse tissues and in the resolution of the complex system of lactate dehydrogenase isozymes including the numerous subbands found in homogenates of mouse tissues. The ontogeny of many enzymes in differentiating tissues from single mouse embryos during postimplantation development can accurately be followed by using this system. After treatment with SDS the same volume of tissue homogenate diluted about five times can also be resolved and stained for protein to give characteristic polypeptide profiles for each tissue.
233
MATERIALS AND METHODS Chemicals. Acrylamide, N,N'-methylenebisacrylamide (Bis), and N , N , N ' , N ' tetramethylenediamine (TEMED) were purchased from Eastman Organic Chemicals Company. Ammonium persulfate, sodium lauryl sulfate (sodium dodecyl sulfate, SDS), Tris base, glycine, Triton X-100, 0003-2697/79/160233-09502.00/09 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
234
OGITA AND MARKERT
Coomassie brilliant blue, and bromophenol blue were obtained from Sigma Chemical Company. Other chemicals were obtained from more than one supplier but all proved to be equally satisfactory. Tissue samples. Tissue samples were obtained from random bred mice (Mus
musculus). Apparatus. The miniaturized electrophoretic apparatus as constructed for use in thin-layer slab gel electrophoresis is shown
A
in Fig. 1. Two slabs may be used at the same time and under identical conditions. The polyacrylamide slabs measure 63 × 72 mm. A frame (Fig. la) is constructed of sheets of Plexiglas 3 mm thick, glued together so as to make a U-shaped frame with outside dimensions 25 × 69 x 90 mm. The glue is made by dissolving Plexiglas in chloroform to make a viscous solution. To the edges of the frame are cemented U-shaped plates of Plexiglas (Ba, B2 in Fig.
.............. ~::~-~L~2.:~ ~
.......
E
FIG. 1. (a) Diagram of the parts, with dimensions, used in the construction of the electrophoresis apparatus. Parts A, B1, and B2 are made from Plexiglas. C1 and C~ are Kodak glass plates. (b) Diagram of the assembled apparatus showing the neoprene gasket (F1) and the removable glass plate (E0 that are clamped to the apparatus to make the gel molds. See text for description of assembly procedure.
MINIATURIZED SYSTEM GEL ELECTROPHORESIS
1), and the inner glass plates of the gel molds (C1, C2) are then permanently affixed to the Plexiglas plates by means of silicgne cement. A leak-free chamber (D), to accOmmodate the inner buffer solution, thus !results (Fig. lb). Another glass plate (El), of the same size as the first, serves as the outer cover of each gel mold (E~, the cover of the second gel mold, is not shown in the figure). We have found Kodak projector slide coVer glasses (3.25 x 4 in. or 83 x 102 mm) to be quite satisfactory for these glass plates. The inner and outer glass plates of each gel mold are separated by neoprene or Plexiglas shims (F), 0.8 mm thick, applied to the sides and bottom of the inner glass plate. The outer plate is pushed against the shims and oriented so as to extend 10 mm above the inner plate. Only the outer glass plate and shims that separate it from the inner plate are removable. They are clamped to the inner plate and supporting Lucite frame to form the molds for casting the gels. Finally, the gel mold is made liquid-tight by dripping melted 2.0% agar around the outside edges. One of the narrow ends of the frame extends 23 mm above the edge of the frame and a 10-mm hole, drilled in it near the top, serves to receive the negative electrode holder. A platinum wire, connected to the holder, extends into the center of the chamber (D) when the apparatus is assembled. The two broad sides of the chamber extend below the narrow ends 25 mm to provide supporting legs when the apparatus is placed upright in the anodal electrode buffer tray (Fig. 2) during electrophoresis. Gradient device. In preparing negative exponential gradients a 12-ml glass vial is used as the mixing chamber and a plastic syringe (10 ml) is used as the reservoir. A thin rubber stopper closes the vial. A disposable hypodermic needle (20 gauge) attached to a plastic syringe (10 ml) is inserted through the stopper until the tip of the needle reaches the middle of the vial. A small magnetic stirrer is used to mix the contents of the vial during the formation
235
" FrG. 2. Photograph of assembled apparatus prior to clamping on the outer glass plates to make the gel molds. Inner and outer electrode chambers are electrically connected through the gel when the inner chamber is filled with buffer.
of the gradient. The mixed acrylamide solution is vented from the mixing vial via a second 20-gauge needle inserted through the rubber stopper and connected to a plastic tube that extends to one side of the bottom of the vial. The upper end of this needle is connected by polyethylene tubing (Intra Medic. PE 100, i.d. 0.034 in., o.d. 0.060 in.) to an applicator for conducting the gel solution into, the gel molds. The applicator is a thin (5 mm) rubber wafer 12 mm in diameter. A 20-gauge hypodermic needle extends 18 mm through the wafer. A metal clip inserted in the wafer to one side of the needle serves to hold the wafer on the lip of the gel mold while the acrylamide solution is conducted into the gel mold. The mixing vial is positioned 70 cm above the gel mold to
236
OGITA AND MARKERT TABLE 1--(Continued)
TABLE 1 STOCK SOLUTIONS USED IN ELECTROPHORESIS
I. Stock Solutions for Running Gel IA: Acrylamide-bis solution Acrylamide Bis Glycerol H20 to make IB: Buffer solutions IBI: Tris-HC1 buffer (0.75 M Tris) Tris HC1 (conc) H20 to make IB2: Tris-HC1-Triton buffer (3.0 M Tris) Tris HC1 (conc) 20% Triton X-100 H20 to make IB3: Tris-HC1-SDS buffer (1,5 M Tris) Tris HC1 (conc) 10% SDS solution H20 to make
39.0 1.0 20.0 100.0
g g ml ml
0.4 g 100.0 ml
IID: 2.0% (v/v) TEMED solution TEMED H~O to make
2.0 ml 100.0 ml
III. Electrode Buffers (pH 8.3)
9.15 g - 3 . 0 ml 100.0 ml
IIIA: Tris-glycine buffer (0.0125 M Tris) Tris Glycine H~O to make
36.6 -12.0 20.0 100.0
g ml ml ml
IIIB: T r i s - H C L - T r i t o n buffer (0.05 M Tris) Tris Glycine 20% Triton-X H~O to make
18.3 -6.0 4.0 100.0
g ml ml ml
IIIC: Tris-glycine-SDS buffer (0.025 M Tris) Tris 3.0 g Glycine 14.4 g 10% SDS 10.0 ml I-I~O to make 1.0 liter
IC: 0.2% (w/v) APS solution Ammonium persulfate H20 to make ID: 0,4% (v/v) TEMED solution TEMED H20 to make
0.2 g 100.0 ml 0.4 ml 100.0 ml
II. Stock Solutions for Stacking Gel IIA: Acrylamide-bis solution Acrylamide Bis Glycerol H20 to make
IIC: 0.4% (w/v) APS solution Ammonium persulfate H20 to make
38.0 2.0 20.0 100.0
liB: Buffer solutions IIBI: Tris-HC1 buffer (0.125 M Tris) Tris 1.5 HC1 (conc) -1.0 H20 to make 100.0 IIB2: Tris-HC1-Triton buffer (0.50 M Tris) Tris 6.0 HC1 (conc) -4.0 20% Triton X-100 20.0 HzO to make 100.0 IIB3: Tris-HC1-SDS buffer (0.25 M Tris) Tris 3.0 HC1 (conc) ~2.0 10% SDS 4.0 HzO to make 100.0
1.5 g 7.2 g 1.0 liter 6.0 28.8 50.0 1.0
g g ml liter
IV. Stock Solutions Used in Sample Preparation IVA: Sample diluting solution (without detergents) 0.5 M Tris-HCl buffer (pH 6,8) 25.0 ml Glycerin 40.0 ml 0.01% BPB 20.0 ml H20 15.0 ml
g g ml ml
IVB: Sample diluting solution with Triton X-100 0.5 M Tris-HC1 buffer (pH 6.8) 25.0 20% Triton X-100 solution 10.0 Glycerin 40.0 0.01% Pyronine Y 20.0 H~O 5.0
g ml ml
IVC: 2.5% SDS solution for treating samples 0.5 M Tris-HC1 buffer (pH 6.8) 3.0 ml 10% SDS solution 5.0 ml Glycerin 10.0 ml fl-mercaptoethanol 2.0 ml
g ml ml ml g ml ml ml
IVD: Diluting solution (after SDS treatment) 0.5 M Tris-HC1 buffer (pH 6.8) 10% SDS Glycerin 0.005% BPB H20
1.5 2.5 5.0 4.0 7.0
ml ml ml ml ml
ml ml ml ml ml
IVE: Bromophenol blue solution (0.005%)
1% BPB methanol H20
0.5 ml 100.0 ml
MINIATURIZED SYSTEM GEL ELECTROPHORESIS
provide the proper flow speed of the acrylamide into the mold.
Composition of stock solutions and buffers. See Table 1. Rinsing the gel molds. To improve the quality of gradient gels the gel molds were rinsed before making the gradient with a solution consisting of 2.5 ml of stock solution IC, 2.5 ml of ID, and 15 ml of H20. When SDS is used in the electrophoresis system, the gel molds are rinsed with a solution composed of 2.5 ml of IC, 2.5 ml of ID, 0.2 ml of a 10% SDS solution, and 14.8 ml of H20. When Triton X-100 is used, the rinsing solution is composed as follows: 2.5 ml IC, 2.5 ml 1D, 1 ml of 20% Triton solution, and 14 ml of H~O. Composition of the running gels. Stock solutions of IA, IB, IC, and H20 are mixed in the proportions as given in Table 1. The proportions of the IA solution to water are varied to give different gel concentrations up to 20% acrylamide gel (Table 2). If higher concentrations of the gel are needed, then IB, IC, and ID stock solutions with twofold higher concentrations should be used for the gel mixture. The mixture is degassed and the ID solution added. When the temperature of the stock solution is the same as the room temperature, degassing is not necessary. The more concentrated polyacrylamide solution is poured into the mixing vial; the less concentrated solution is placed in the upper plastic syringe. Preparation of the running gel. The gel is typically made with a continuous 20 to 8% exponential gradient. The gradient is made using 2 ml o f the 20% acrylamide solution in the mixing vial and 5 ml of 8% acrylamide in the upper plastic syringe. A small stirring bar is placed in the mixing vial. The mixture is gently stirred and withdrawn at a rate of about 0.8 to 1 ml per minute through a siphon of polyethylene tubing (i.d. 0.034 in., o.d. 0.060 in.) which is inserted via an applicator into the gel mold. The volume of the gel solution remains constant at 2 ml in the mixing vial and is continuously diluted
237
by the incoming solution from the upper syringe. The gel solution is poured into the mold until it reaches a level 10 mm from the top of the mold. It is then gently overlayered with water by using a 19-gauge hypodermic needle connected by plastic tubing to a plastic syringe filled with water. The gel is allowed to polymerize for 40 min; then the water and any residual unpolymerized gel mixture are completely removed with a piece of filter paper from the mold chamber above the upper edge of the running gel. The gel may be stored at this stage overnight by covering the top of the gel with a fourfold dilution of the IB gel buffer solution. Preparation of the stacking gel. The stacking gel mixture is prepared by mixing the solutions as given in Table 2. A 4% acrylamide stacking gel is optimal for nearly all experiments. Prior to addition of the IID solution, the stacking gel solution is degassed. The buffer in the gel mold on top of the running gel is carefully removed and water is poured into the upper electrode chamber to a level 10 mm below the top of the gel mold. Then the stacking gel mixture is pipetted into the upper part of the mold on top of the running gel. To form the sample slots in the gel, a comb is inserted into the stacking gel solution before the stacking gel solution begins to polymerize so as to reach within 2 to 3 mm of the upper surface of the running gel. The comb is made from an 0.8-mm-thick Teflon sheet, which is slightly thinner than the gel mold, so that the comb can be removed easily after the gel has set. Then enough water is gently poured into the upper electrode chamber to flow across the top of the gel mold and gently layer over the stacking gel. The stacking gel is allowed to polymerize for about 20 min. After gelation, all water is removed from the electrode chamber. The bottom shim and agar which seals the bottom of the gel mold are also removed. Then the apparatus is placed upright in the anodal electrode buffer tray which is already filled with the electrode
238
OGITA AND MARKERT TABLE 2 COMPOSITION OF POLYACRYLAMIDE GELS OF DIFFERENT CONCENTRATIONS
Composition of stacking gels a
Composition of running gels~
Percentage gel
Stock solution IA (ml)
I-I20 (ml)
Percentage gel
Stock solution IIA (ml)
H~O (ml)
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
3.0 3.5 4.0 4.5 5.0 5.5 6.0
1.50 1.75 2.00 2.25 2.50 2.75 3.00
8.50 8.25 8.00 7.75 7.50 7.25 7.00
a In preparing each concentration of the gel according to these tables, aliquots of solutions IB, IC, and ID are also added in the ratio of 5 ml o f l B , 2.5 ml o f l C , and 2.5 ml o f l D .
buffer. Next, the electrode buffer is gently poured into the cathode chamber until it fills the chamber and covers the top of the stacking gel. Removal of the Teflon comb must be done slowly so as to leave the sample wells intact. Finally, any residual unpolymerized gel mixture is completely removed from each sample slot by injecting a stream of air bubbles in buffer into the slot with a syringe. The sample slots remain filled with buffer which will be displaced by the heavier samples (mixed with glycerin) when they are pipetted onto the bottom of the slots. Preparation of tissue extracts. Tissues were either frozen in a Revco freezer at -70°C or used fresh. Tissues were generally homogenized in twice their weight of distilled water using a Potter-Elvejhem homogenizer. For small samples, such as tissues obtained from single mouse embryos, the tissues were placed in small plastic centri-
fuge tubes (Evergreen Scientific Company, Catalog No. 3014) with twice their weight of distilled water. Such samples were homogenized by a motor-driven plastic pestle. Homogenates were then centrifuged at 20,000g for 60 min at 4°C to remove cell debris. After centrifugation, any lipid layer is aspirated off and discarded and the clear supernatant is used immediately or stored in 1.5-ml Eppendorf plastic vials at -70°C. Very small samples, such as a single mouse egg are placed in small plastic centrifuge tubes with 5/zl of IVB solution, frozen, and thawed (three to five cycles), and centrifuged at 10,000g for 5 min at 4°C. The supernatant is then introduced into a sample slot in the gel. Sample preparations: (1) Tris-HCl system and Triton X-IO0 system. Preparation of samples to be used in the Tris-HC1 system without detergents requires mixing 10
MINIATURIZED SYSTEM GEL ELECTROPHORESIS
/zl of the extract with 10/zl of solution IVA. In the Triton X-100 system 10/zl of extract is mixed with I0 tzl of solution IVB. Sample preparations: (2) SDS system. In preparing protein samples for the SDS gel system 2 mg of a dry sample is dissolved in 1.0 ml of the IVC SDS solution. The solution is then placed in a boiling water bath for 5 min, cooled, and diluted with an equal aliquot of water. Then a further dilution is made 4:1 with stock solution IVE. After this the concentration of the protein in the sample solution is adjusted with the IVD sample diluting solution, if necessary, so that the final protein concentration is approximately 1 /xg//zl. Usually 3/~1 of solution is loaded into each slot in the gel. In the case of wet tissue, the tissue is lysed in the IVC solution in the ratio 1 mg of wet tissue to 5 /zl of the IVC solution by heating in boiling water for 5 min. Any remaining insoluble materials are removed by pipet and discarded. The solution is reheated for 2 min and diluted with an equal volume of water. The solution is then further diluted 4:1 with the IVE solution. In the case of tissue extracts, an aliquot of the IVC solution is added to the extract and this solution is heated in a boiling water bath for 5 min. The solution is then diluted 4:1 with IVE solution. After centrifugation at 20,000g for 20 min, the supernatants are used immediately or stored in sealed vials at -20°C until used. Before use, the samples are again heated to 100°C for 2 rain by immersing the vials in boiling water. After cooling, 3-tzl aliquots of the samples are pipetted into the sample wells in the gel. Staining procedures. Staining procedures for enzymes and proteins on gels have been described by many investigators. Those described here were modified as appropriate for this particular gel system. After electrophoresis, the apparatus is placed in a horizontal position with the outer glass plate of the gel mold on top. This glass plate is carefully removed by using a thin spatula to pry between the neoprene or plastic shims on
239
the sides and the top glass plate. After removal of the outside sealing agar, the glass plate and the two shims are removed, leaving only the acrylamide gel slab adhering to the inner glass plate of the gel mold. The gel slab can then be removed under a gentle stream of water with a spatula and transferred to a staining solution. Protein staining. Investigators commonly fix the SDS proteins in the gel slab with 50% trichloroacetic acid (TCA) overnight before staining. However, we have found that immediate staining for 1 h at 25°C with a 0.1% Coomassie brilliant blue solution freshly made up in 50% TCA is quite satisfactory, and prefixing in TCA is unnecessary. The gels were usually destained by repeated washing in 10% acetic acid for approximately 48 h. However, the stained gel slab may be heated in 10% acetic acid at 60°C to achieve total destaining in approximately 2 h.
Staining procedures for undenatured proteins. Immediately after electrophoresis the gel slab is placed in a staining solution consisting of 0.5 g of amid0 black 10B in 25 ml of methanol, 5 ml of acetic acid, and 22.5 ml of deionized water. After 30 rain in the staining solution the stained gels are transferred to 10% acetic acid and heated at 60°C for 2 h. All the dye not bound to protein washes out of the gel. By using this method for protein staining, the proteins are simultaneously fixed and stained. Esterase staining. After electrophoresis the gels are washed in three changes of 0.01 M phosphate buffer at pH 6.8 for 10 rain each. Then the gels are incubated in a reaction mixture consisting of 50 ml of 0.05 M phosphate buffer at pH 6.8, 1 ml of 1% anapthyl acetate in acetone solution, and 200 mg of fast violet B salt for 30 rain at 37°C. After incubation the stained gels are washed in running water. Lactate dehydrogenase staining. Lactate dehydrogenase (LDH) isozymes were revealed by staining with a mixture containing 66 mg nicotinamide adenine dinu-
240
OGITA AND MARKERT
cleotide (NAD), 35 mg nitroblue tetrazolium (NBT), 2 mg p h e n a z i n e m e t h o s u l f a t e (PMS), 10 ml of 1 N sodium lactate solution, 10 ml of 10% Triton X-100 aqueous solution, 50 ml of 0.1 M T r i s - H C 1 buffer (pH 7.0), and 30 ml of deionized water. The gel slab was incubated for 30 min at 37°C in the staining solution and then washed in running water to remove any remaining reactants.
4-
RESULTS AND DISCUSSION Our miniaturized e]ectrophoresis apparatus is simple and economical to assemble and use. It is also easy to disassemble the gel molds for cleaning. The fact that two gels can be run at the same time under the same conditions makes accurate comparison possible. In addition the fiat gel, in contrast to disc or cylindrical gels, can hold as many as 12 samples side by side for ready comparison. The thin gels and narrow sample slots make the apparatus suitable for analyzing very small samples. Homogenates contain-. ing about 10/xg of protein can be resolved into 50 to 100 distinct bands of polypeptides by SDS electrophoresis (Fig. 3). In undenatured preparations many isozymes of a variety of enzymes can easily be identified. More than 30 esterases from mouse tissues
m
1 2 3 4 5 6 7 8
FIG. 4. Photograph of esterase zymogram of mouse tissues and organs. More than 30 bands are evident. Each organ has a characteristic pattern of esterases. Channel 1--serum; 2--colon; 3--ileum; 4--jejunum; 5--duodenum; 6--stomach; 7--liver; 8--kidney.
were observed, for example, after staining with a-naphthyl acetate as substrate and fast violet B salt as dye coupler (Fig. 4). The many subbands of the L D H isozymes of mouse tissues were readily resolved (Fig. 5). The molecular basis of these subbands
-lall~m
n
m
ee - B~.
i ilii!!ii!i~!i
U m
C~.-!
1
3456789101112
FIG. 3. Photograph of gel showingresolution of proteins from mouse organ homogenates after SDS treatment. Each channel was loaded with 1 to 3/~g of protein. Channel 1- - heart; 2- - harderian gland; 3--lungs; 4--brain; 5--pancreas; 6--kidney; 7--epididymis; 8--testis; 9--liver; 10--salivary glands; ll--thymus; 12--stomach.
- A~
~iii!
12345
678910
FIG. 5. Photograph of lactate dehydrogenase zymogram showing the five principal isozymes and the numerous subbands of several of the isozymes. The sperm-specific isozyme (C4) is also evident. Channel 1- - salivary gland; 2- - kidney; 3-- testis; 4-- intestine; 5--liver; 6- - brain; 7-- eye; 8- - diaphragm; 9-- skeletal muscle; 10--heart.
MINIATURIZED SYSTEM GEL ELECTROPHORESIS
has been analyzed and the results published (7). The structure of the electrophoresis apparatus insures adequate cooling when electrophoresis is carried out in a cold room at 5°C. No additional cooling is needed. One side of the gel mold is in direct contact with the buffer in the cathode chamber and the other side is exposed to air. This arrangement proves adequate to remove heat generated in the thin gels during electrophoresis. We find that a constant current of 10 to 20 mA (with both gel molds in use) provides good separation of proteins in about 2 to 4 h. The voltage usually varies from 80 to 340 V during the running of the gel, but this does not appreciably affect the patterns of resolved proteins. The gels made in this apparatus are fiat, thin, and clear and have smooth surfaces and uniform thickness. After staining for protein or enzymes the gels may be readily scanned with a suitable densitometer to obtain a semiquantitative measure of the amount of protein or enzyme in each band. We found that the Shimadzu dual-wavelength tic scanner (CS-910) equipped with a U-225 MCS recorder and ChromatopacE1A printing data processor for calculating the areas of each peak was particularly satisfactory for obtaining densitometric profiles. However, densitometric scanning of zymograms provides only a rough indication of relative enzyme activities. By running a series of dilutions under rigorously standardized conditions an accurate measure of
241
enzyme activity can be obtained and used in analyzing and comparing isozyme profiles obtained with different tissue homogenates. The use of Triton X-100 in the gels releases bound enzymes and proteins in the sample, which otherwise would remain at the origin. However, the most rapidly migrating bands may be retarded somewhat by the detergent. Triton also makes handling the gel easier as during removal from the mold. Bromophenol blue is not suitable as a dye marker for the buffer front in the gel when Triton is used because it complexes with the Triton. In operation, when fast violet B (esterase staining) and NBT (LDH staining) were used, Triton increased the amount of dye deposited in zymograms and thus enhanced the sensitivity of the detection system. The mechanism of Triton action is complex and not yet worked out in detail. REFERENCES 1. Raymond, S., and Weintraub, L. (1959) Science 130, 711. 2. Shapiro, A. L., Vinuela, E., and Maizel, J. V., Jr. (1967)Biochem. Biophys. Res. Commun. 28, 815-820. 3. Weber, K., and Osborn, M. J. (1969)J. Biol. Chem. 244, 4406-4412. 4. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 5. O'Farrell, P. H. (1975)J. Biol. Chem. 250, 40074021. 6. Scheele, G. A. (1975)J. Biol. Chem. 250, 53755385. 7. Yamamura, K.-I., Ogita, Z.-I., and Markert, C. L. (1979) J. Exp. Zool. 208, 271-280.
A Miniaturized System for Electrophoresis on Polyacrylamide Gels t,2 Z E N - I C H I OGITA* AND C L E M E N T L . MARKERT'~
*Research Institute for Oriental Medicines, University of Toyama, Gofuku, Toyama, 930 Japan, and tDepartment of Biology, Yale Unif~ersity, New Haven, Connecticut 06520 Received May 29, 1979 The design, construction, and use of a simple economical system for gel electrophoresis are described, along with effective procedures and the required materials. Examples of successful use of the system are presented.
Polyacrylamide gel electrophoresis, since its introduction in 1959 by Raymond and Weintraub (1), has proved to be a useful and powerful method for separating proteins and nucleic acids. The system has been extended for the estimation of molecular weights of polypeptide chains by using the anionic detergent, sodium dodecyl sulfate (SDS) 3 (2-4). Recently O'Farrell (5) and Scheele (6) have described a two-dimensional polyacrylamide gel electrophoretic technique that effectively resolves complex protein mixtures obtained from tissue homogenates. However, we have not found these methods as published to be satisfactory for resolving and measuring enzymes at very low concentrations. Therefore, we developed an improved miniaturized system with greater sensitivity and high resolving power. 1 This work was supported in part by a Scientific Research Grant of the Ministry of Education of Japan, by a Grant of the Ministry of Health and Welfare of Japan, and by NIH Biomedical Research Support grant 5-S07-RR-07015. 2 This paper is dedicated to the memory of Dr. Alvin Nason. 3 Abbreviations used: SDS, sodium dodecyl sulfate; Bis, N,N'-methylenebisacrylamide; TEMED, N, N,N',N'-tetramethylenediamine; TCA, trichloroacetic acid; LDH, lactate dehydrogenase; NBT, nitroblue tetrazo|ium; PMS, phenazine methosulfate; tlc, thin-layer chromatography; BPB, bromophenol blue.
In this paper we describe this miniaturized polyacrylamide vertical slab gel electrophoretic system. It is simple to construct and economical, resolves 3-/A samples of protein solutions, and is a considerable improvement on many polyacrylamide systems now in common use. We have tested the system in the analysis of the esterases in homogenates of mouse tissues and in the resolution of the complex system of lactate dehydrogenase isozymes including the numerous subbands found in homogenates of mouse tissues. The ontogeny of many enzymes in differentiating tissues from single mouse embryos during postimplantation development can accurately be followed by using this system. After treatment with SDS the same volume of tissue homogenate diluted about five times can also be resolved and stained for protein to give characteristic polypeptide profiles for each tissue.
233
MATERIALS AND METHODS Chemicals. Acrylamide, N,N'-methylenebisacrylamide (Bis), and N , N , N ' , N ' tetramethylenediamine (TEMED) were purchased from Eastman Organic Chemicals Company. Ammonium persulfate, sodium lauryl sulfate (sodium dodecyl sulfate, SDS), Tris base, glycine, Triton X-100, 0003-2697/79/160233-09502.00/09 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
234
OGITA AND MARKERT
Coomassie brilliant blue, and bromophenol blue were obtained from Sigma Chemical Company. Other chemicals were obtained from more than one supplier but all proved to be equally satisfactory. Tissue samples. Tissue samples were obtained from random bred mice (Mus
musculus). Apparatus. The miniaturized electrophoretic apparatus as constructed for use in thin-layer slab gel electrophoresis is shown
A
in Fig. 1. Two slabs may be used at the same time and under identical conditions. The polyacrylamide slabs measure 63 × 72 mm. A frame (Fig. la) is constructed of sheets of Plexiglas 3 mm thick, glued together so as to make a U-shaped frame with outside dimensions 25 × 69 x 90 mm. The glue is made by dissolving Plexiglas in chloroform to make a viscous solution. To the edges of the frame are cemented U-shaped plates of Plexiglas (Ba, B2 in Fig.
.............. ~::~-~L~2.:~ ~
.......
E
FIG. 1. (a) Diagram of the parts, with dimensions, used in the construction of the electrophoresis apparatus. Parts A, B1, and B2 are made from Plexiglas. C1 and C~ are Kodak glass plates. (b) Diagram of the assembled apparatus showing the neoprene gasket (F1) and the removable glass plate (E0 that are clamped to the apparatus to make the gel molds. See text for description of assembly procedure.
MINIATURIZED SYSTEM GEL ELECTROPHORESIS
1), and the inner glass plates of the gel molds (C1, C2) are then permanently affixed to the Plexiglas plates by means of silicgne cement. A leak-free chamber (D), to accOmmodate the inner buffer solution, thus !results (Fig. lb). Another glass plate (El), of the same size as the first, serves as the outer cover of each gel mold (E~, the cover of the second gel mold, is not shown in the figure). We have found Kodak projector slide coVer glasses (3.25 x 4 in. or 83 x 102 mm) to be quite satisfactory for these glass plates. The inner and outer glass plates of each gel mold are separated by neoprene or Plexiglas shims (F), 0.8 mm thick, applied to the sides and bottom of the inner glass plate. The outer plate is pushed against the shims and oriented so as to extend 10 mm above the inner plate. Only the outer glass plate and shims that separate it from the inner plate are removable. They are clamped to the inner plate and supporting Lucite frame to form the molds for casting the gels. Finally, the gel mold is made liquid-tight by dripping melted 2.0% agar around the outside edges. One of the narrow ends of the frame extends 23 mm above the edge of the frame and a 10-mm hole, drilled in it near the top, serves to receive the negative electrode holder. A platinum wire, connected to the holder, extends into the center of the chamber (D) when the apparatus is assembled. The two broad sides of the chamber extend below the narrow ends 25 mm to provide supporting legs when the apparatus is placed upright in the anodal electrode buffer tray (Fig. 2) during electrophoresis. Gradient device. In preparing negative exponential gradients a 12-ml glass vial is used as the mixing chamber and a plastic syringe (10 ml) is used as the reservoir. A thin rubber stopper closes the vial. A disposable hypodermic needle (20 gauge) attached to a plastic syringe (10 ml) is inserted through the stopper until the tip of the needle reaches the middle of the vial. A small magnetic stirrer is used to mix the contents of the vial during the formation
235
" FrG. 2. Photograph of assembled apparatus prior to clamping on the outer glass plates to make the gel molds. Inner and outer electrode chambers are electrically connected through the gel when the inner chamber is filled with buffer.
of the gradient. The mixed acrylamide solution is vented from the mixing vial via a second 20-gauge needle inserted through the rubber stopper and connected to a plastic tube that extends to one side of the bottom of the vial. The upper end of this needle is connected by polyethylene tubing (Intra Medic. PE 100, i.d. 0.034 in., o.d. 0.060 in.) to an applicator for conducting the gel solution into, the gel molds. The applicator is a thin (5 mm) rubber wafer 12 mm in diameter. A 20-gauge hypodermic needle extends 18 mm through the wafer. A metal clip inserted in the wafer to one side of the needle serves to hold the wafer on the lip of the gel mold while the acrylamide solution is conducted into the gel mold. The mixing vial is positioned 70 cm above the gel mold to
236
OGITA AND MARKERT TABLE 1--(Continued)
TABLE 1 STOCK SOLUTIONS USED IN ELECTROPHORESIS
I. Stock Solutions for Running Gel IA: Acrylamide-bis solution Acrylamide Bis Glycerol H20 to make IB: Buffer solutions IBI: Tris-HC1 buffer (0.75 M Tris) Tris HC1 (conc) H20 to make IB2: Tris-HC1-Triton buffer (3.0 M Tris) Tris HC1 (conc) 20% Triton X-100 H20 to make IB3: Tris-HC1-SDS buffer (1,5 M Tris) Tris HC1 (conc) 10% SDS solution H20 to make
39.0 1.0 20.0 100.0
g g ml ml
0.4 g 100.0 ml
IID: 2.0% (v/v) TEMED solution TEMED H~O to make
2.0 ml 100.0 ml
III. Electrode Buffers (pH 8.3)
9.15 g - 3 . 0 ml 100.0 ml
IIIA: Tris-glycine buffer (0.0125 M Tris) Tris Glycine H~O to make
36.6 -12.0 20.0 100.0
g ml ml ml
IIIB: T r i s - H C L - T r i t o n buffer (0.05 M Tris) Tris Glycine 20% Triton-X H~O to make
18.3 -6.0 4.0 100.0
g ml ml ml
IIIC: Tris-glycine-SDS buffer (0.025 M Tris) Tris 3.0 g Glycine 14.4 g 10% SDS 10.0 ml I-I~O to make 1.0 liter
IC: 0.2% (w/v) APS solution Ammonium persulfate H20 to make ID: 0,4% (v/v) TEMED solution TEMED H20 to make
0.2 g 100.0 ml 0.4 ml 100.0 ml
II. Stock Solutions for Stacking Gel IIA: Acrylamide-bis solution Acrylamide Bis Glycerol H20 to make
IIC: 0.4% (w/v) APS solution Ammonium persulfate H20 to make
38.0 2.0 20.0 100.0
liB: Buffer solutions IIBI: Tris-HC1 buffer (0.125 M Tris) Tris 1.5 HC1 (conc) -1.0 H20 to make 100.0 IIB2: Tris-HC1-Triton buffer (0.50 M Tris) Tris 6.0 HC1 (conc) -4.0 20% Triton X-100 20.0 HzO to make 100.0 IIB3: Tris-HC1-SDS buffer (0.25 M Tris) Tris 3.0 HC1 (conc) ~2.0 10% SDS 4.0 HzO to make 100.0
1.5 g 7.2 g 1.0 liter 6.0 28.8 50.0 1.0
g g ml liter
IV. Stock Solutions Used in Sample Preparation IVA: Sample diluting solution (without detergents) 0.5 M Tris-HCl buffer (pH 6,8) 25.0 ml Glycerin 40.0 ml 0.01% BPB 20.0 ml H20 15.0 ml
g g ml ml
IVB: Sample diluting solution with Triton X-100 0.5 M Tris-HC1 buffer (pH 6.8) 25.0 20% Triton X-100 solution 10.0 Glycerin 40.0 0.01% Pyronine Y 20.0 H~O 5.0
g ml ml
IVC: 2.5% SDS solution for treating samples 0.5 M Tris-HC1 buffer (pH 6.8) 3.0 ml 10% SDS solution 5.0 ml Glycerin 10.0 ml fl-mercaptoethanol 2.0 ml
g ml ml ml g ml ml ml
IVD: Diluting solution (after SDS treatment) 0.5 M Tris-HC1 buffer (pH 6.8) 10% SDS Glycerin 0.005% BPB H20
1.5 2.5 5.0 4.0 7.0
ml ml ml ml ml
ml ml ml ml ml
IVE: Bromophenol blue solution (0.005%)
1% BPB methanol H20
0.5 ml 100.0 ml
MINIATURIZED SYSTEM GEL ELECTROPHORESIS
provide the proper flow speed of the acrylamide into the mold.
Composition of stock solutions and buffers. See Table 1. Rinsing the gel molds. To improve the quality of gradient gels the gel molds were rinsed before making the gradient with a solution consisting of 2.5 ml of stock solution IC, 2.5 ml of ID, and 15 ml of H20. When SDS is used in the electrophoresis system, the gel molds are rinsed with a solution composed of 2.5 ml of IC, 2.5 ml of ID, 0.2 ml of a 10% SDS solution, and 14.8 ml of H20. When Triton X-100 is used, the rinsing solution is composed as follows: 2.5 ml IC, 2.5 ml 1D, 1 ml of 20% Triton solution, and 14 ml of H~O. Composition of the running gels. Stock solutions of IA, IB, IC, and H20 are mixed in the proportions as given in Table 1. The proportions of the IA solution to water are varied to give different gel concentrations up to 20% acrylamide gel (Table 2). If higher concentrations of the gel are needed, then IB, IC, and ID stock solutions with twofold higher concentrations should be used for the gel mixture. The mixture is degassed and the ID solution added. When the temperature of the stock solution is the same as the room temperature, degassing is not necessary. The more concentrated polyacrylamide solution is poured into the mixing vial; the less concentrated solution is placed in the upper plastic syringe. Preparation of the running gel. The gel is typically made with a continuous 20 to 8% exponential gradient. The gradient is made using 2 ml o f the 20% acrylamide solution in the mixing vial and 5 ml of 8% acrylamide in the upper plastic syringe. A small stirring bar is placed in the mixing vial. The mixture is gently stirred and withdrawn at a rate of about 0.8 to 1 ml per minute through a siphon of polyethylene tubing (i.d. 0.034 in., o.d. 0.060 in.) which is inserted via an applicator into the gel mold. The volume of the gel solution remains constant at 2 ml in the mixing vial and is continuously diluted
237
by the incoming solution from the upper syringe. The gel solution is poured into the mold until it reaches a level 10 mm from the top of the mold. It is then gently overlayered with water by using a 19-gauge hypodermic needle connected by plastic tubing to a plastic syringe filled with water. The gel is allowed to polymerize for 40 min; then the water and any residual unpolymerized gel mixture are completely removed with a piece of filter paper from the mold chamber above the upper edge of the running gel. The gel may be stored at this stage overnight by covering the top of the gel with a fourfold dilution of the IB gel buffer solution. Preparation of the stacking gel. The stacking gel mixture is prepared by mixing the solutions as given in Table 2. A 4% acrylamide stacking gel is optimal for nearly all experiments. Prior to addition of the IID solution, the stacking gel solution is degassed. The buffer in the gel mold on top of the running gel is carefully removed and water is poured into the upper electrode chamber to a level 10 mm below the top of the gel mold. Then the stacking gel mixture is pipetted into the upper part of the mold on top of the running gel. To form the sample slots in the gel, a comb is inserted into the stacking gel solution before the stacking gel solution begins to polymerize so as to reach within 2 to 3 mm of the upper surface of the running gel. The comb is made from an 0.8-mm-thick Teflon sheet, which is slightly thinner than the gel mold, so that the comb can be removed easily after the gel has set. Then enough water is gently poured into the upper electrode chamber to flow across the top of the gel mold and gently layer over the stacking gel. The stacking gel is allowed to polymerize for about 20 min. After gelation, all water is removed from the electrode chamber. The bottom shim and agar which seals the bottom of the gel mold are also removed. Then the apparatus is placed upright in the anodal electrode buffer tray which is already filled with the electrode
238
OGITA AND MARKERT TABLE 2 COMPOSITION OF POLYACRYLAMIDE GELS OF DIFFERENT CONCENTRATIONS
Composition of stacking gels a
Composition of running gels~
Percentage gel
Stock solution IA (ml)
I-I20 (ml)
Percentage gel
Stock solution IIA (ml)
H~O (ml)
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
3.0 3.5 4.0 4.5 5.0 5.5 6.0
1.50 1.75 2.00 2.25 2.50 2.75 3.00
8.50 8.25 8.00 7.75 7.50 7.25 7.00
a In preparing each concentration of the gel according to these tables, aliquots of solutions IB, IC, and ID are also added in the ratio of 5 ml o f l B , 2.5 ml o f l C , and 2.5 ml o f l D .
buffer. Next, the electrode buffer is gently poured into the cathode chamber until it fills the chamber and covers the top of the stacking gel. Removal of the Teflon comb must be done slowly so as to leave the sample wells intact. Finally, any residual unpolymerized gel mixture is completely removed from each sample slot by injecting a stream of air bubbles in buffer into the slot with a syringe. The sample slots remain filled with buffer which will be displaced by the heavier samples (mixed with glycerin) when they are pipetted onto the bottom of the slots. Preparation of tissue extracts. Tissues were either frozen in a Revco freezer at -70°C or used fresh. Tissues were generally homogenized in twice their weight of distilled water using a Potter-Elvejhem homogenizer. For small samples, such as tissues obtained from single mouse embryos, the tissues were placed in small plastic centri-
fuge tubes (Evergreen Scientific Company, Catalog No. 3014) with twice their weight of distilled water. Such samples were homogenized by a motor-driven plastic pestle. Homogenates were then centrifuged at 20,000g for 60 min at 4°C to remove cell debris. After centrifugation, any lipid layer is aspirated off and discarded and the clear supernatant is used immediately or stored in 1.5-ml Eppendorf plastic vials at -70°C. Very small samples, such as a single mouse egg are placed in small plastic centrifuge tubes with 5/zl of IVB solution, frozen, and thawed (three to five cycles), and centrifuged at 10,000g for 5 min at 4°C. The supernatant is then introduced into a sample slot in the gel. Sample preparations: (1) Tris-HCl system and Triton X-IO0 system. Preparation of samples to be used in the Tris-HC1 system without detergents requires mixing 10
MINIATURIZED SYSTEM GEL ELECTROPHORESIS
/zl of the extract with 10/zl of solution IVA. In the Triton X-100 system 10/zl of extract is mixed with I0 tzl of solution IVB. Sample preparations: (2) SDS system. In preparing protein samples for the SDS gel system 2 mg of a dry sample is dissolved in 1.0 ml of the IVC SDS solution. The solution is then placed in a boiling water bath for 5 min, cooled, and diluted with an equal aliquot of water. Then a further dilution is made 4:1 with stock solution IVE. After this the concentration of the protein in the sample solution is adjusted with the IVD sample diluting solution, if necessary, so that the final protein concentration is approximately 1 /xg//zl. Usually 3/~1 of solution is loaded into each slot in the gel. In the case of wet tissue, the tissue is lysed in the IVC solution in the ratio 1 mg of wet tissue to 5 /zl of the IVC solution by heating in boiling water for 5 min. Any remaining insoluble materials are removed by pipet and discarded. The solution is reheated for 2 min and diluted with an equal volume of water. The solution is then further diluted 4:1 with the IVE solution. In the case of tissue extracts, an aliquot of the IVC solution is added to the extract and this solution is heated in a boiling water bath for 5 min. The solution is then diluted 4:1 with IVE solution. After centrifugation at 20,000g for 20 min, the supernatants are used immediately or stored in sealed vials at -20°C until used. Before use, the samples are again heated to 100°C for 2 rain by immersing the vials in boiling water. After cooling, 3-tzl aliquots of the samples are pipetted into the sample wells in the gel. Staining procedures. Staining procedures for enzymes and proteins on gels have been described by many investigators. Those described here were modified as appropriate for this particular gel system. After electrophoresis, the apparatus is placed in a horizontal position with the outer glass plate of the gel mold on top. This glass plate is carefully removed by using a thin spatula to pry between the neoprene or plastic shims on
239
the sides and the top glass plate. After removal of the outside sealing agar, the glass plate and the two shims are removed, leaving only the acrylamide gel slab adhering to the inner glass plate of the gel mold. The gel slab can then be removed under a gentle stream of water with a spatula and transferred to a staining solution. Protein staining. Investigators commonly fix the SDS proteins in the gel slab with 50% trichloroacetic acid (TCA) overnight before staining. However, we have found that immediate staining for 1 h at 25°C with a 0.1% Coomassie brilliant blue solution freshly made up in 50% TCA is quite satisfactory, and prefixing in TCA is unnecessary. The gels were usually destained by repeated washing in 10% acetic acid for approximately 48 h. However, the stained gel slab may be heated in 10% acetic acid at 60°C to achieve total destaining in approximately 2 h.
Staining procedures for undenatured proteins. Immediately after electrophoresis the gel slab is placed in a staining solution consisting of 0.5 g of amid0 black 10B in 25 ml of methanol, 5 ml of acetic acid, and 22.5 ml of deionized water. After 30 rain in the staining solution the stained gels are transferred to 10% acetic acid and heated at 60°C for 2 h. All the dye not bound to protein washes out of the gel. By using this method for protein staining, the proteins are simultaneously fixed and stained. Esterase staining. After electrophoresis the gels are washed in three changes of 0.01 M phosphate buffer at pH 6.8 for 10 rain each. Then the gels are incubated in a reaction mixture consisting of 50 ml of 0.05 M phosphate buffer at pH 6.8, 1 ml of 1% anapthyl acetate in acetone solution, and 200 mg of fast violet B salt for 30 rain at 37°C. After incubation the stained gels are washed in running water. Lactate dehydrogenase staining. Lactate dehydrogenase (LDH) isozymes were revealed by staining with a mixture containing 66 mg nicotinamide adenine dinu-
240
OGITA AND MARKERT
cleotide (NAD), 35 mg nitroblue tetrazolium (NBT), 2 mg p h e n a z i n e m e t h o s u l f a t e (PMS), 10 ml of 1 N sodium lactate solution, 10 ml of 10% Triton X-100 aqueous solution, 50 ml of 0.1 M T r i s - H C 1 buffer (pH 7.0), and 30 ml of deionized water. The gel slab was incubated for 30 min at 37°C in the staining solution and then washed in running water to remove any remaining reactants.
4-
RESULTS AND DISCUSSION Our miniaturized e]ectrophoresis apparatus is simple and economical to assemble and use. It is also easy to disassemble the gel molds for cleaning. The fact that two gels can be run at the same time under the same conditions makes accurate comparison possible. In addition the fiat gel, in contrast to disc or cylindrical gels, can hold as many as 12 samples side by side for ready comparison. The thin gels and narrow sample slots make the apparatus suitable for analyzing very small samples. Homogenates contain-. ing about 10/xg of protein can be resolved into 50 to 100 distinct bands of polypeptides by SDS electrophoresis (Fig. 3). In undenatured preparations many isozymes of a variety of enzymes can easily be identified. More than 30 esterases from mouse tissues
m
1 2 3 4 5 6 7 8
FIG. 4. Photograph of esterase zymogram of mouse tissues and organs. More than 30 bands are evident. Each organ has a characteristic pattern of esterases. Channel 1--serum; 2--colon; 3--ileum; 4--jejunum; 5--duodenum; 6--stomach; 7--liver; 8--kidney.
were observed, for example, after staining with a-naphthyl acetate as substrate and fast violet B salt as dye coupler (Fig. 4). The many subbands of the L D H isozymes of mouse tissues were readily resolved (Fig. 5). The molecular basis of these subbands
-lall~m
n
m
ee - B~.
i ilii!!ii!i~!i
U m
C~.-!
1
3456789101112
FIG. 3. Photograph of gel showingresolution of proteins from mouse organ homogenates after SDS treatment. Each channel was loaded with 1 to 3/~g of protein. Channel 1- - heart; 2- - harderian gland; 3--lungs; 4--brain; 5--pancreas; 6--kidney; 7--epididymis; 8--testis; 9--liver; 10--salivary glands; ll--thymus; 12--stomach.
- A~
~iii!
12345
678910
FIG. 5. Photograph of lactate dehydrogenase zymogram showing the five principal isozymes and the numerous subbands of several of the isozymes. The sperm-specific isozyme (C4) is also evident. Channel 1- - salivary gland; 2- - kidney; 3-- testis; 4-- intestine; 5--liver; 6- - brain; 7-- eye; 8- - diaphragm; 9-- skeletal muscle; 10--heart.
MINIATURIZED SYSTEM GEL ELECTROPHORESIS
has been analyzed and the results published (7). The structure of the electrophoresis apparatus insures adequate cooling when electrophoresis is carried out in a cold room at 5°C. No additional cooling is needed. One side of the gel mold is in direct contact with the buffer in the cathode chamber and the other side is exposed to air. This arrangement proves adequate to remove heat generated in the thin gels during electrophoresis. We find that a constant current of 10 to 20 mA (with both gel molds in use) provides good separation of proteins in about 2 to 4 h. The voltage usually varies from 80 to 340 V during the running of the gel, but this does not appreciably affect the patterns of resolved proteins. The gels made in this apparatus are fiat, thin, and clear and have smooth surfaces and uniform thickness. After staining for protein or enzymes the gels may be readily scanned with a suitable densitometer to obtain a semiquantitative measure of the amount of protein or enzyme in each band. We found that the Shimadzu dual-wavelength tic scanner (CS-910) equipped with a U-225 MCS recorder and ChromatopacE1A printing data processor for calculating the areas of each peak was particularly satisfactory for obtaining densitometric profiles. However, densitometric scanning of zymograms provides only a rough indication of relative enzyme activities. By running a series of dilutions under rigorously standardized conditions an accurate measure of
241
enzyme activity can be obtained and used in analyzing and comparing isozyme profiles obtained with different tissue homogenates. The use of Triton X-100 in the gels releases bound enzymes and proteins in the sample, which otherwise would remain at the origin. However, the most rapidly migrating bands may be retarded somewhat by the detergent. Triton also makes handling the gel easier as during removal from the mold. Bromophenol blue is not suitable as a dye marker for the buffer front in the gel when Triton is used because it complexes with the Triton. In operation, when fast violet B (esterase staining) and NBT (LDH staining) were used, Triton increased the amount of dye deposited in zymograms and thus enhanced the sensitivity of the detection system. The mechanism of Triton action is complex and not yet worked out in detail. REFERENCES 1. Raymond, S., and Weintraub, L. (1959) Science 130, 711. 2. Shapiro, A. L., Vinuela, E., and Maizel, J. V., Jr. (1967)Biochem. Biophys. Res. Commun. 28, 815-820. 3. Weber, K., and Osborn, M. J. (1969)J. Biol. Chem. 244, 4406-4412. 4. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 5. O'Farrell, P. H. (1975)J. Biol. Chem. 250, 40074021. 6. Scheele, G. A. (1975)J. Biol. Chem. 250, 53755385. 7. Yamamura, K.-I., Ogita, Z.-I., and Markert, C. L. (1979) J. Exp. Zool. 208, 271-280.
