ANALYTICAL BIOCHEMISTRY 45, 19%201 (1972)

General Aspects of Analytical Isotac,hophoresis

of Proteins in Polyacrylamide Gels

ANN GRIFFITH

Departsment of Anatomy, University of Zllinois at the Medical Center, Chicago, Zllinois 60612

AND

NICHOLAS CATSIMPOOLAS

Laboratory of Protein Chemistry, Central Soya Research Center, Chicago, Illinois 60639

Received May 12, 1971

Isotachophoresis is an electrophoretic method of separation of ion species exhibiting the same sign of charge (negative or positive), and all having a common counterion. At equilibrium, all ions move with the same migration velocity. The ionic species to be separated are spaced between a leading and a terminating ion. The principle, historical development, and some applications of the technique were reviewed recently by Hag- lund (1). Isotachophoresis can be applied to the separation of proteins if Ampholine carrier ampholytes (2) are used as “spacers,” that is, as intermediate-mobility compounds which can force two adjacent protein zones apart. Svendsen and Rose (3) used a preparative vertical column- electrophoresis apparatus to separate isotachophoretically human serum proteins in polyacrylamide gel taking advantage of the spacer technique. They noted that the possible variations of the experimental parameters offer significant versatility in protein separations.

The present report examines the feasibility, limitations, and some general factors involved in the isotachophoretic separation of proteins in polyacrylamide gel columns similar to those used in disc electro- phoresis (4) and gel isoelectrofocusing (5). The results suggest that isotachophoresis will emerge as a new analytical tool capable of unique separations by intelligent planning of experimental conditions.

192 @ 1972 by Academic Press, Inc.

ANALYTICAL GEL ISOTACHOPHORESIS 193

EXPERIMENTAL

Reagents

Acrylamide, N,N,N’,N’-tetramethylethylenediamine (TEMED) ,I and riboflavin were purchased from Canalco, Rockville, Md. N,N’-Methyl- enebisacrylamide (Bis), 2- amino-a- (hydroxymethyl) -1,3-propanediol (Tris), and glycine were purchased from Eastman Kodak Co., Rochester, N. Y. Sucrose was purchased from Merck & Co., Rahway, N. J. Mercuric chloride was obtained from J. T. Baker, Chemical Co., Phillipsburg, N. J., and bromophenol blue indicator from Hartman-Leddon Co., Philadelphia, Pa. The carrier ampholytes were obtained from LKB- Produkter, Bromma, Sweden.

Test Materials

Bovine serum albumin (BSA) (tryst.) was obtained from Pentex, Kankakee, Ill. Hemoglobin (BHb) (bovine) (2 X tryst.) and soybean trypsin inhibitor (STI) were purchased from Mann Research Labora- tories, New York, N. Y.

Gel Formulations and Experimental Procedure

Gel were prepared by photopolymerization (4) of acrylamide using the buffers described by Svendscn and Rose (3). The stock acrylamide solution contained 30 gm acrylamicle and 1 gm Bis, and was made up to 100 ml with water. The stock catalyst solution was prepared by dis- solving 4 mg riboflavin in 100 ml water.

The pH 4.0 gel buffer solution consisted of 0.9 gm Tris, 3 ml glacial acetic acid, and 0.3 ml TEMED, and was made up to 100 ml with water. The pH 4.5 gel buffer contained 2 gm Tris, 3 ml glacial acetic acid, and 0.3 ml TEMED, and was made up to 100 ml with water. The pH 6.1 gel buffer consisted of 5.2 gm Tris, 39 ml 1 h’i H,PO,, and 0.5 ml TEMED. and was made up to 100 ml with water.

The stock sucrose solution was 25% in water. The terminal electrolyte (pH 8.2 buffer) consisted of 6 gm Tris and

30 gm glycine in 2000 ml water. The protein solutions of appropriate concentration were prepared by

dissolving the test materials in the terminal electrolyte buffer (pH 8.2). The gel solutions were prepared by mixing 3 ml each of the stock

acrylamide solution, stock catalyst solution, gel buffer of appropriate pH (4.0, 4.5. or 6.1), and the st,ock sucroic solution. 12 ml of water was

1 Abbreviations: TEMED. N,N.N’.N’-tctramethylenediamine; Bis, N,N’-methyl- enebisacrylnmide ; Tris. 2-amino-2-(hydroxgmethyl)-1,3-propanediol; BSA, bovine serum albumin ; RHb, bovine hemoglobin ; STI, soybean trypsin inhibitor.

194 GRIFFITH AND CATSIMPOOLAS

added to 12 ml of the above mixture to produce gels of 3.75% acrylamide concentration. To produce 5% gels, 6 ml of water was added to 12 ml of the above gel mixture. The gel solution was used to fill 5 mm i.d. glass tubes to a height of 6 cm. The mixtures were layered with the terminal electrolyte buffer to produce a flat surface and were photopolymerized for 1 hr with fluorescent light. The gel columns were turned at 30 min to ensure even polymerization. After polymerization, a mixture of carrier ampholytesZ of the appropriate pH range, protein solution, and sucrose solution was layered between the gel-terminating buffer interface. Iso- tachophoresis was then performed in a Shandon electrophoresis apparatus with both the upper and the lower baths filled with the terminal electro- lyte buffer (pH 8.2). The upper bath was connected to the cathode. Con- stant current (5 mA/gel column) was supplied by a Shandon Vokam power supply.

After isotachophoresis the gels were removed from the glass columns and placed in a mercuric bromophenol blue staining solution for 15 min. Destaining was accomplished overnight in a 30% ethyl alcohol/5% glacial acetic acid solution. The gels are stored in 7.5% acetic acid. The staining method is a modification of the procedure of Awdeh (6) and prevents fading of the color of the bands. The stain is prepared by dis- solving 50 gm mercuric chloride and 0.5 gm bromophenol blue in 50% ethyl alcohol to a total volume of 500 m1.3 Another advantage of this staining method is that the location of the Ampholine in the gel can be observed. Directly after staining and before destaining, the gel area con- taining the ampholyte stains yellow. The areas of the gel not containing the ampholyte appear reddish. Other staining procedures suitable for gels containing carrier ampholytes (7) can be used in isotachophoresis gels.

RESULTS

Preliminary Consideratiorbs

In the present studies, glycinate has been chosen as the terminating ion, whereas acetate (pH 4.0 and pH 4.5 gel buffers) and phosphate (pH 6.1 gel buffer) are the leading ions. The carrier ampholytes act as spacers of intermediate mobility between the protein zones to be separated. Tris is the common counterion in the system. It should be realized that other terminating and leading ions can be used and, of course, different results can be expected. The protein sample has been mixed with the carrier ampholytes and applied on top of the gel for convenience. Al- ternatively, the carrier ampholytes can be incorporated into the upper

* 40% stock solution, ‘Avoid contact with skin.

ANALYTICAL GEL ISOTACHOPHORESIS 195

part of the gel column before polymerization (3). However, carrier am- pholytes of certain pH range may inhibit polymerization in the absence of ammonium persulfate catalyst (7).

Three proteins, namely, bovine hemoglobin (BHb), bovine serum albumin (BSA), and soybean trypsin inhibitor (STI), alone or in a mixture, were used for these experiments. According to their isoelectric points, these proteins should migrate isotachophoretically toward the positive electrode in the order: soybean trypsin inhibitor, bovine serum albumin, and bovine hemoglobin. The size of the protein does not con- tribute to isotachophoretic migration provided the polyacrylamide gel concentration is sufficiently low to avoid sieving.

Effect of pH Range of Carrier Ampholytes

In the experiment illustrated in Fig. 1 (upper), the pH of the gel buffer (6.1) and concentration of carrier ampholytes were kept constant. However, ampholytes covering the pH ranges 3 to 6, and 3 to 10, re- spect.ively, were used for comparative purposes. In the pH 3-10 range, all three proteins (STI, BSA, and BHb) entered the gel with sufficient migration in 20 min. Minor components in both the BHb and ST1 sam- ples can be seen. In the mixture, the major components were separated but the minor ones were overlapping. In the pH 3-6 range, the BHb sample barely entered the gel, but the distance of separation from the BSA sample was greater than in the pH 3-10 range. In the pH 3-6 range, all three proteins did not migrate as far into the gel as in the pH 3-10 range. Similar results are shown in Fig. 1 (lower), using pH 4.5 gel buffer and a different time of isotachophoresis. Thus, differences in the migration behavior of these proteins in different pH range carrier ampholytes was observed as can be expected if the ampholytes act as spacers.

Effect of Carrier Ampholyte Concentration

A mixture of BHb, BSA, and ST1 was mixed with increasing amounts of pH 3-6 carrier ampholytes and subjected to isotachophoresis in pH 4.5 gels for 60 min. The effect of carrier ampholyte concentration was dramatic (Fig. 2, upper). With increasing ampholyte concentration, pro- tein band separation was increased. In very small concentrations, the ampholyte seems to be located in a narrow band in the center of the gel ; this may account for the lower degree of separation noted in these gels. At high ampholyte concentrations, the proteins did not enter the gels during the 60 min of electrophoresis. The same phenomenon was observed even after isotachophoresis for 90 min using pH 3-6 ampholytes and pH 4.5 gel buffer (Fig. 2, lower). Thus, the amount of carrier ampho- lyte present in the sample appears to be critical not only for efficient zone

196 GRIFFITH AND CATSIMPOOLAS

FIG. 1. Effect of pH range of Ampholine on gel isotachophoresis. Upper: gel buffer pH 6.1; time 20 min; current 5 mA/column; gel concentration 3.75%; (A) BHb (66 Fg) and 25 ~1 pH 3-10 Ampholine; (B) BSA (66 pg) and 25 pl pH 3-10 Ampholine; (C) ST1 (66 ag) and 25 pl pH 3-10 Ampholine: (D) mix- ture of BHb, BSA, and ST1 (66 pg each) and 25 ~1 pH 3-10 Ampholine; (E) BHb (66 fig) and 25 pl pH 3-6 Ampholine; (F) BSA (66 pg) and 25 pl pH 3-6 Ampholine; (G) ST1 (66 pg) and 25 ~1 pH 3-6 Ampholine; (H) mixture of BHb, BSA, and STI (66 fig each) and 25 pl pH 36 Ampholine. Lower: gel buffer pH 4.5; time (A through D) 45 min and (E through G) 90 min; current 5 mA/column; gel concentration 3.75%; (A) BHb (500 pg) and 50 pl pH 3-6 Ampholine; (B) BSA (250 cg) and 50 pl pH 3-6 Ampholine; (C) STI (250 ag) and 50 pl pH 3-6 Ampholine; (D) mixture of BHb (500 pg) and BSA (250 pg)

with 50 ~1 pH 3-6 Ampholine; (E) BHb (150 pg) and 50 pl pH 3-10 Ampholine; (F) BSA (150 pg) and 50 pl pH 3-10 Ampholine; (G) ST1 (150 pg) and 50 pl pH 3-10 Ampholine. Migration is toward the anode (top of photograph).

ANALYTICAL GEL ISOTACHOPHORESIS 197

c 00 A : @ 0 0

FIG. 2. Effect of Ampholine present in sample on gel isotachophoresis of proteins. Upper: gel buffer pH 4.5; time 60 min; current 5 mA/column; gel concentration 3.75%; protein sample, mixture of 66 fig each of BHb. BSA, and ST1 in all gels; pH 3-6 Ampholine, 12 pl (A), 25 pl (B), 50 pl (C), 100 pl (D), and 200 pl (E). Lozoer: gel buffer pH 4.5; time 90 min; current 5 mA/column; gel concentration 3.75%; pH 3-6; Ampholine (A) BHb (150 ag) and 25 pl Ampholine; (B) BHb (150 pg) and 50 ~1 Ampholine; (C) BHb (150 pg) and 100 ~1 Ampholine; (D) BSA (150 pg) and 25 ~1 Ampholine; (E) BSA (150 Fg) and 100 ~1 Ampholine ; (F) BSA (150 pg) and 200 pl Ampholine; (G) mixture of BHb and BSA (150 gg each) and 50 ,pl Ampholine; (H) mixture of BHb and BSA (150 pg each) and 100 81 Ampholine. Migration is toward the anode (top of photograph).

198 GRIFFITH AND CATSIMPOOLAS

separation but also for the appearance of the proteins in the gel within a certain time of isotachophoresis.

Effect of Protein Concentration

Svendsen and Rose (3) stated that “the distance by which two peaks are set apart is approximately proportional to the amount of Ampholine added per unit of protein amount to be separated.” Our results, shown in Fig. 3, demonstrate that the distance between two protein zones does depend on the ratio of Ampholine to protein only to a minor extent. Large differences depend on the actual amount of the Ampholine in the sample, as illustrated in Fig. 2. Varying the protein concentration and keeping the amount of Ampholine constant (Fig. 3) produced only small differences in the isotachophoresis patterns.

FIG. 3. Effect of protein concentration on gel isotachophoresis: gel buffer pH 4.5; time 60 min; current 5 mA/column; gel concentration 3.75%; pH 3-6 Ampho- line (25 $1 in each gel). Protein sample, mixture of BHb, BSA, and STI of the fol- lowing amounts each: 15 pg (A), 30 gg (B), 45 gg (C), 60 gg (D), 75 pg (E), 90 pg W), 105 pg (G), 120 ,ug (HI.

Effect of pH of Gel Buffer

In order to evaluate the effect of the gel buffer, isotachophoresis was performed for 60 min using pH 3-6 Ampholine and varying the pH of the gel buffer. Three different gel buffers were used, of pH 4.0, 4.5, and 6.1. In the pH 4.0 and pH 4.5 buffers, the leading ion is acetate whereas, in the pH 6.1 buffer, the leading ion is phosphate. The terminating ion

ANALYTICAL GEL ISO’PACHOPHORESIS 199

Ra. 4. Effect of changing pEf of leading electrolyte buffer on gel isotachophoresis: pH 36 Ampholine (25 ~1 in each sample) ; time 60 min ; current 5 mA/column; gel concentration 3.75%. Protein sample in A, B, and C, mixture of BHb, BSA, and ST1 (67 pg each) ; in D, BSA (66 pg) ; in E, ST1 (66 pg) ; and in F, BHb (66 pg). Gel buffer, pH 4.0 (A), pH 4.5 (B), pH 6.1 (C), pH 4.0 (D), pH 4.5 (E), and pH 6.1 (F). Gel in F is inverted (upside down in the picture).

again is glycinate and the common counterion is Tris. The results are shown in Fig. 4. It appeared that the higher the pII of the gel buffer, the faster was the migration of the proteins and Ampholine. With the pro- teins under study, better resoiution was obtained with the pH 4.0 buffer, BSA being separated into two major components.

Effect of Acrylamide Concentration

In gel isotachophoresis experiments similar to gel isoelectrofocusing, the ae~lamide concentration should be as low as possible to avoid “sieving effects.” Resistance to isotachophoretic migration is demonstrated in Fig. 5 by using 5.0% polyacrylamide gels in an experiment similar to that illustrated in Fig. 4 with 3.75% gels. It may be seen that the mi- gration of BSA and BHb has been retarded. The ST1 being a molecule of small size (XIW a~pro~i~~3at~ly 22,000) has migrated faster and prob- ably isotachophoretically. Thus, the acrylamide concentration can be increased for special separations but should not be above 4% for general isotaehophoresis experiments. It should be realized that, for certain proteins of large size, even 5% gels may offer resistance to migration,

200 GRIFFITH AND ~ATS~~~OOLAS

l?m. 5. Effect of acryiamide concentration (gel porosity) on gel isotachophoresis: pH 3-6 Ampholine (25 pf in each sample) ; time 60 min; current 5 mA/column; gel concentration 5.0%. Protein sample in A, B, and C, mixture of BHb, BSA, and ST1 (66 gg each) ; in D, BHb (66 fig) ; in E and H, BSA (66 pg) ; and in I? and G, ST1 (66 pg). Gel buffer, pH 4.0 (A), pH 4.5 (B), pH 6.1 (0, pH 4.0 (D, E, and F), and pH 4.5 (G and El). The isotachophoresis patterns in this figure (5% gel) should be compared with similar patterns in Fig. 4 (3.75% gel) to visualize dif- ferences in migration.

DISCUSSION

The data presented in this paper indicate that separation of proteins by analytical gel isotachophoresis using carrier ampholytes as “spacers” is affected by a variety of experimental factors. Some of these parameters involve the pH range of the carrier ampholytes used, the actual amount of carrier ampholyte present in the sample, the pH of the gel buffer and the nature of the leading ion, the time at which isotaehophoresis is terminated, and the acrylamide concentration in the gels. Undoubtedly, it should be expected that also the pH and nature of the terminating electrolyte, which was not examined in this work, may affect the separa- tion achieved. Other factors may involve the nature of the counterion, the current intensity, temperature of the run, and to some extent the ratio of carrier ampholytes to protein present in the sample.

Because of the variety of experimental factors, the technique offers versatility in designing specific separations. The analytical method may save time, effort: and expenses in defining the best conditions for prep- arative purposes. However, the authors do not recommend that gel isotachophoresis be used as a “one-shot” experiment in obtaining the “pattern” of an unknown mixture as can be done with disc electrophoresis (4) and to a lesser extent with gel isoelectrofocusing (7). In this respect, gel isotachophoresis is uniquely different in comparison with the above-

ANALYTICAI, (;EL ISOTACIIOPIIOIZESlS 201

mentioned electrophoretic techniques. As Svendsen and Rose (3) have shown, excellent separations can he nrhicved by sp&matic change of conditions.

The major drawback of gel isotachophoresis is that separations may have to be monitored at different time intervals. Protein zones may have been separated at such a distance that they do not appear simultaneously in the gel at a given time of electrophoresis. In situ scanning techniques such as developed for gel isoelectrofocusing (8) may be very helpful in this respect, and will allow one to take full advantage of the capabilities of the met’hod. This problem can also be partly solved by using very long polyacrylamide gels.

Haglund (1) has pointed out certain advantages that isotachophoresis of proteins using carrier ampholytes can offer over isoelectrofocusing. In isotachophoresis, the carrier ampholytes form a ‘(moving pH gradient” with the proteins migrating more or less according to their pI values. However, the proteins have a charge at all times so that insolubility problems such as occur in isoelectrofocusing can be avoided. In our esti- mation, both techniques have certain advantages and disadvantages de- pending on the problem at hand. Some excellent separations have been achieved by isoelectrofocusing and it is hoped that isotachophoresis will

alleviate some of the drawbacks of this method.

SUMMARY

Separations of proteins in polyacrylamide gels on a micro scale can be achieved by isotachophoresis using carrier ampholytes as “spacers.” Some of the general factors that affect the migration and separation of protein zones involve the pH range of Ampholine, the amount of Ampholine present in the sample, the pH of the gel buffer (containing the leading ion), the time of isotachophoresis, and gel porosity. Other factors that may affect isotachophoresis of protein using “spacers” are discussed.

ACKNOWLEDGMENT

This research was supported in part by National Institutes of Mental Health Grant No. 8396.

REFERENCES

1. H. HAGLUND, Sci. Took- 17, 2 (1970). 2. 0. VESTERBERG AND H. SVENSSON, Acta Chem. &and. 20, 820 (1966) 3. P. J. SVENDSEN AND C. ROSE, Sci. Took 17, 13 (1970). 4. B. J. DAVIS, Ann. N. Y. Acnd. Sci. 121, 464 (1964). 5. N. CATSIMPOOLAS, Anal. Biochem. 26, 480 (1968). 6. Z. L. AWDEH, Sci. Took 16, 42 (1969). 7. N. CATSIMPOOLAS, Sepnr. fki. 5, 523 (1970). 8. N. CATSIMPO~LAS AND J. WANG, Aml. Biochsm. 39, 141 (1971).