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Isoelectric focusing in polyacrylamide-agarose

Isoelectric focusing in polyacrylamide-agarose

ANALYTICAL BIOCHEMISTRY 139,77-8 1 (1984) Isoelectric Focusing in Polyacrylamide-Agarose’ RICHARDM.PINOANDTIMOTHYK.HART Department of Anatomy. L...

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139,77-8 1 (1984)



in Polyacrylamide-Agarose’

RICHARDM.PINOANDTIMOTHYK.HART Department of Anatomy. Louisiana State University Medical Center, Medical Education Building, 1901 Perdido Street, New Orleans, Louisiana 70112 Received September 19, 1983 A procedure using a 2.5% acrylamide-0.5% agarose gel for slab or tube isoelectric focusing is described. This composite gel is durable and enables a rapid focusing of high-molecular-weight compounds. KEY WORDS: isoelectric focusing; polyacrylamide; agarose; macromolecules; composite gel.

such as agarose-Sephadex (4). This composite gel demonstrates virtually no electroendosmotic effects (4), but requires focusing times of 5 h for 3-mm-thick gels (4) and at least I.5 h for l-mm gels (Pharmacia Fine Chemicals, personal communication). In the present report we describe the use of a stable polyacrylamide-agarose gel for rapid isoelectric focusing analysis in tube gels and slabs.

Analytical isoelectric focusing has been traditionally performed in polyacrylamide (1) or agarose gels (2). Polyacrylamide gels offer a high degree of mechanical strength, which enables the use of high field strengths and power for excellent resolution but are not suitable for large molecule analysis due to molecular sieving effects. Large molecules may be focused relatively unhindered in agarose (2). Electroendosmotic effects, one drawback of this medium (3), have been largely eliminated with the advent of commercially available agaroses with low electroendosmotic properties (2), although these effects still have been reported to occur (4). In addition, agarose gels must be used within a short time after preparation to obtain reproducible electrophoretic results (2), cannot be used in tube gels, and often exhibit a thinning of the gel near the anode during prolonged focusing (5). Uriel and Berges (6) described the use of composite polyacrylamide-agarose gels for electrophoresis. These are strong, easy to handle, and can be used at acrylamide concentrations less than 5%. Because of the low monomer content the electrophoresis of highmolecular-weight molecules is facilitated (6,7). Isoelectric focusing in composite gels has been limited to media designed for preparative use,



Slab Gel Method Gelpreparation. Composite gels containing 2.5% polyacrylamide-0.5% agarose-2% ampholyte were prepared as follows. For 20 ml of gel mixture 1.0 ml ampholyte (BioLyte 3110; Bio-Rad Laboratories, Richmond, Calif.), 4.0 ml 25% (w/v) glycerin, 3.0 ml distilled-deionized water, and 2.0 ml 25% acrylamide-0.75% bisacrylamide were mixed, degassed (5- 10 min), and placed in a 60°C water bath. To this solution 10 ml 1% agarose (IsoGel Agarose, FMC Corp., Rockland, Maine; Zero-m, Agarose, Bio-Rad) kept at 60°C were added followed by 50 ~1 each of 0.1% riboflavin-5’-monophosphate and 2% ammonium persulfate (Bio-Rad) or 2.5 ~1 of 10% ammonium persulfate alone. For the isoelectric focusing of lectins, 0.2 h4 galactose

’ Supported by the National Institutes of Health Grant EY 03776. 77

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(Sigma Chemical Co., St. Louis, MO.) was added to the mixture. Slab gels were prepared using a Bio-Rad capillary thin-layer casting tray. The tray and 45 X IOO- or 125 X 100-mm glass plates were warmed by rinsing in hot water. The hydrophobic side of GelBond (FMC) was applied to the glass plates with water and placed face down (hydrophilic surface) on the 0.8 mm thickness side of the casting tray. The gel mixture was applied between the tray and hydrophilic surface with a warmed pipet, and the agarose in the mixture was allowed to solidify (lo-20 min) prior to the polymerization of the acrylamide by fluorescent light if riboflavin was used. After polymerization (1 h with riboflavin; 30 min with ammonium persulfate alone), gels adherent to the GelBond were removed from the tray and used immediately, or covered with Parafilm and stored in sealed plastic bags at 4°C. Focusing conditions. Slab gel focusing was done on a Bio-Rad 14 15 apparatus cooled to 4°C. Electrodes were placed on filter paper wicks resting on the gel, with 1 N NaOH used as the catholyte and 0.5 N acetic acid as the anolyte. Samples were placed directly on the gel, on filter paper applicators, or in wells l-2 cm from the cathode. The samples were run into the gels at 30-50 V/cm or 0.08 W/ cm2 for 10 min. Excess sample liquid was then removed. If the samples were placed in wells, at this point the wells were filled with 1% agarose-2% ampholyte. Alternatively, gels were prefocused at 30-50 V/cm for 25 min prior to sample application. Focusing was accomplished using a constant power of 0.15-0.30 W/cm’ with a limit of 1200- 1500 V. Migration rate determination. The migration rate in polyacrylamide-agarose of a large protein was compared to rates obtained in polyacrylamide and agarose gels. Native ferritin was chosen as the test protein since it is visible in gels during focusing, has a molecular weight of 480,000, and bears a p1 of 3.8-4.4. Polyacrylamide-agarose slab gels were prepared as described above. Polyacrylamide gels

(4%T, 4%C) were mounted on GelBond PAG. Agarose ( 1% IsoGel) gels containing 10% sorbitol were mounted on GelBond. All gels measured 0.8 X 75 X 110 mm and contained 2% Bio-Lyte 3/10 ampholyte. Sodium hydroxide (1 N) and 0.5 N acetic acid served as catholyte and anolyte, respectively. Gels were prefocused at 40 V/cm for 25 min. Measurements with a surface pH electrode indicated that pH gradients were established by this time in all gels. Native ferritin (20 ~1; 2.0 mg) and 20 ~1 IsoGel p1 markers (FMC Corp.; diluted 1:2 with distilled water) were applied to the prefocused gels via filter paper applicators placed 1.O cm from the cathode. Samples were run into the gels at 40 V/cm (3 min) and the filter paper applicators were removed. Isoelectric focusing in the three gel types was completed at 100 V/cm. Ferritin migration rates in polyacrylamide-agarose and agarose gels at 150 V/cm were also examined. During focusing the distance from the cathode of the visible, advancing ferritin band was measured as a function of time. These measurements were compared to the position of pI markers after focusing was completed. Fixation, staining, and @al processing. Two methods were used to stain slab gels. (i) Gels were fixed for 1 h in 4% sulfosalicylic acid and 12.5% trichloroacetic acid, and stained in 0.04% Coomassie blue R 250-0.05% crocein scarlet in 25% ethanol- 10% acetic acid containing 0.05% CuS04 (l), and destained in i 2% ethanol-7% acetic acid-O.O5% CuSO4, followed by a final rinse in the same solution without CuS04. (ii) Gels were fixed as above or with 3.5% sulfosalicylic acid-5% trichloroacetic acid-5% ZnSO, (8), washed in water for lo-30 min, covered with a layer of No. 577 Schleicher and Schuell (Keene, NH) filter paper, and inverted onto several layers of paper towels, and a l-kg weight was placed on top (2). After 20-30 min the gel was removed from the towels with the filter paper left in place. The gel was then partially dried with a hot-air dryer and the filter paper was lifted off while still damp. Gels were stained with





Coomassie blue-crocein scarlet and destained as described above. Gels fixed in the solution containing ZnS04 were silver stained by the method of Willoughby and Lambert (8) and destained in 7% acetic acid. In the final step, all gels were rinsed in 12% ethanol-7% acetic acid and dried at 60°C or at room temperature. Tube Gel Method Gel preparation. The polyacrylamide-agarose mixture used for slab gels was also used for tube gels. Gel tubes (5 X 130 mm) were warmed to 60°C, their bottoms were covered with Parafilm, and they were placed in a tube gel polymerization stand. The tubes were filled to within 1.5 cm of the top with the composite medium. After 3-5 min the top of the gel was overlayed with 0.1 ml of the sample suspended in the gel mixture, followed by a third layer of gel. After the agarose solidified, the acrylamide was allowed by polymerize by fluores-

cent light (1 h). To prevent the polymerized gels from slipping out of the gel tubes, the bottom of each tube was inserted into dialysis tubing (Spectropor 4, 6 mm diameter; Spectrum Industries, Los Angeles, Calif.) and the open end was sealed. Focusing conditions. Tube gel focusing (at 4°C) was performed using 0.5 N acetic acid as the anolyte and 0.01 N NaOH as the catholyte. Samples were run into the gel using 20 V/cm (15 min). Focusing was done at 70-80 V/cm. Fixation and staining. Gels were fixed ( l-2 h), stained with Coomassie blue-crocein scarlet, destained in 12% ethanol-lo% acetic acid, and stored in fixative. RESULTS AND DISCUSSION

We have employed composite polyacrylamide-agarose gels for isoelectric focusing for 1 year with reproducible results. Figure 1 presents a comparison between the

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FIG. 1. The migration rates of native fenitin in three gel Fenitin focuses faster in the composite gel consisting of V/cm than in 1% agarose at the same field strength (0). composite gel (Cl) was more rapid than in 1% agarose (0)

types used in isoelecbic focusing are compared. 2.5% polyacrylamide-OS% agarose (w) at 150 Similarly, at 100 V/cm the migration in the and 4% polyacrylamide (A).



migration rates of native ferritin in three media under identical conditions. At a field strength of 100 V/cm the migration rate in 2.5% polyacrylamide-0.5% agarose was 0.67 cm/min, which was 34% faster than that observed for 1% agarose (0.5 cm/min) and 67% greater than in polyacrylamide (0.22 cm/min) at a reduced concentration of 4% T. Migration rates at 150 V/cm were 1.4 and 1.O cm/min for composite and agarose gels, respectively; a difference of 40%. At this field strength overheating of the agarose gels was apparent. This rapid focusing in polyacrylamide-agarose is equivalent to that obtainable in ultrathin polyacrylamide gels (9). Protein markers (Fig. 2A) and high-molecular-weight compounds such as I,G-ferritin (Fig. 2B; M, > 630,000) and soybean agglutinin-ferritin (Fig. 2C; M, > 590,000) conjugates were completely focused without sieving on slab gels within 30 min. Tube gel IEF using the composite medium required a short focusing time of 60 min A-

B 4


FIG. 2. Isoelectric focusing on polyacrylamidc-agarose. Arrowheads indicate point of sample application (A-C). Slab gels. (A) IsoGel pI markers, silver stained, (B) I,G-ferritin conjugate, silver stained; (C) soybean agglutinin-ferritin conjugate, Coomassie blue-cm&n scarlet stained, (D) Tube gel. Rabbit IF, Coomassie blue-cm&n scarlet stained.

(Fig. 2D). After 90 min some skewing of bands was evident. Several variations in gel composition, preparation, and staining were examined during the course of this study. The source of agarose was critical. Best results were obtained with IsoGel agarose. No difference was observed between gels used immediately after polymerization or after storage at 4°C in sealed bags for up to 3 weeks. Composite gels did not swell in staining or destaining solutions, as do polyacrylamide gels. If the staining method using partially dried gels was used, the entire process from making the gel to its final dried state took less than 4 h. Zero-m, Agarose (Bio-Rad) also formed sturdy composite gels. However, they did not bind to the GelBond as well as did gels made with IsoGel. With Agarose IEF (Pharmacia, Piscataway, N. J.) the gels did not polymerize. GelBond, a support for agarose, provided the best substrate for the gels. Although the acrylamide content was higher than that of agarose, the gels did not adhere to the polyacrylamide support GelBond PAG (FMC). Compared to 1% agarose gels, the reduced agarose content of the composite gel provided a lower viscosity, which facilitated casting. Silver-stained gels with little to no background were obtained by the method of Willoughby and Lambert (8) designed for agarose gels. It is important to use a staining chamber similar to the one they have described (8) to reduce the surface area of the stain exposed to air in order to reduce background staining rather than to conserve chemicals as the authors have suggested. Silver staining, using a protocol for polyacrylamide gels ( lo), yielded high background staining. For all staining methods for best final gel preparations were obtained by drying the gels at room temperature or at 60°C after destaining. This produced pliable gels virtually fused to the GelBond. Treatment with glycerin-ethanol solutions commonly used for polyacrylamide gels resulted in a sticky gel surface that did not completely dry.




ACKNOWLEDGMENTS The authors thank Dr. L. Wong and Dr. M.-Y. Chuang for their helpful suggestions during the comae of this study.

REFERENCES I. Righetti, P. G., and Drysdale, J. W. ( 1974) J. Chromatogr. 98, 271-32 1. 2. Saravis, C. A,, and Zamcheck, N. (1979) J. Immunol. Methods 29,9 l-96. 3. Righetti, P. G., and Drysdale, J. W. (1976) Isoelectric Focusing, pp. 424-425, Elsevier, New York/Amsterdam. 4. Manrique, A., and Lasky, M. (198 1) Eiectrophoresis 2, 3 15-320.


5. Ebers, G. C., Rice, G. P., and Armstrong, H. (1980) J. Immunol. Methods 31, 3 15-323. 6. Uriel, J., and Berges, J. (1974) in Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel (Allen, R. C., and Mauer, H. R., eds.), pp. 235-245, de Gruyter, New York. 7. Peacock, A. C., and Dingman, C. W. (1968) B&hem. J. I, 668-674. 8. Willoughby, E. W., and Lambert, A. (1983) Anal. Biochem. 130, 353-358. 9. Kinzkofer, A., and Radola, B. J. (198 1) Electrophoresis 2,174-183.

10. Merril, C. R., Goldman, D., Sedman, S. A., and Ebert, M. H. (1981) Science 211, 1437-1438.