ANALYTICAL BIOCHEMISTRY 16, 234-243 (1966)

Improved Methods of Molding Starch Gel for

Zone Electrophoresis

JOJI HORI

From the Chemistry Laboratory, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya, Japan

Received March 7, 1966

Starch gel is a highly suitable supporting medium for high-resolution zone electrophoresis because of its molecular sieving effect. Recently, numerous techniques for eluting protein fractions from the gel or scan- ning electropherogram with a densitometer were reported by many inves- tigators. In these techniques, one of the physical properties of the gel- the difference in migration according to thickness of the gel-constitutes a serious obstacle to analytical estimations obtained, and exerts an un- favorable influence upon resolution and reproducibility of the patterns.

As Baur (1) has pointed out, distortion of the vertical zonal profiles of electrophoresed substrates can be diEregarded to a certain degree by the use of a thin layer of starch gel. Grculade and Ollivier (2) and Matsui and Yaeno (3) succeeded in decreasing the zonal distortion of thin-layer gels by employing their original me1 hotis of gel molding. However, the structure of the gel molds employed is m both cases intricate, the opera- tional procedure is complicated, al.11 in addition satisfactory removal of the zonal distortion was found to be difficult.

This report describes two methods of molding the starch gel medium by which the zonal distortion during electrophoresis can be practically removed. The methods resemble somewhat those of Groulade and Matsui, but may be applicable to the preparation of the original thick-layer gel as well as the thin-layer gel, and are recommended for obtaining exact estimations in qualitative or quantitative analysis for developed protein components.

MATERIALS AND METHODS

The starch grain used was prepared by hydrolyzing the commercial starch grain of Japanese potato (Hoei Ind. Co.) at 385°C. The 15.5% gel was prepared with borate buffer pH 8.78 (containing 0.03 M boric acid and 0.012 M sodium hydroxide).

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MOLDING METHODS FOR STARCH GEL 235

Elect8rophorcsis of human serum was conducted either by the hori- zontal or the vertical method, by keeping a gel tray (see below for description) horizontal or vertical. The samples are inserted into the gel by the usual technique (4) (in liquid form, in starch grain wicks, or in filter paper strips). Contact between gel and buffer contained in a vessel on either side of the gel is made by a “membrane bridge” (5). With the vertical method, the double-stage type of apparatus described pre- viously (5) was also used. In this apparatus, the bottom of the gel tray was fixed to the cooling plate for circulating cold water.

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FIG. 1. Construction of gel mold for preparation of thick-layer gel by Method I: (A) shallow dish; (B) gel mold; (C) gel tray; (a) removable wedge for slot forma- tion; (b) plastic cover; (c,d,e) plastic frames; (f) sheet of Saran Wrap; (g) glass plate.

Molding Method I. Figure 1 shows the design of the gel mold for prepa- ration of the original thick-layer gel. A hard glass plate (about 1 mm in thickness) wetted with distilled water is placed horizontally, and on it :L sheet of Saran Wrap* is smoothed out. Over the Saran is spread out further a sheet of filter paper, and firm pressure is then applied evenly to the filter paper with a rubber roller. The free ends of the Saran projecting from the plate are folded beneath the plate. and a shallow dish (Fig. 1A) is assembled by placing three identic:llly shaped Plexiglas

’ Asahi-Dow Limited, Japan.

236 JOJI HOE1

frames, each 3 mm thick, on top of each other on the Saran. After pouring the warm gel into this dish, two cover plates are placed into position, and a removable wedge for slot formation is introduced into the gel as shown in Fig. 1B. Superfluous gel is pressed out from the gel mold thus assembled either by clamping the edges of the mold or by applying firm pressure on the cover plates. After gelation is completed, the removable wedge for slot formation, the cover plates, and the topmost frame are removed one by one from the mold, in which the gel is 3 mm higher than the fixed side wall. The excess gel is cut off by moving horizontally a cutting blade supported on both longitudinal sides of the middle frame of the mold. The tray containing a gel block 6 mm thick (Fig. 1C) is thus made.

For assembling the gel mold for either the thin-layer or the micro method, two frames, each with a thickness of 1.5 or 2 mm, are used in place of the foregoing three frames. After thorough cooling of the gel in the mold, the upper frame is removed from the mold and excess gel is cut off as in t.he preceding method, permitting the preparation of a gel layer 1.5 or 2 mm thick.

When the filter paper method of sample insertion is used, the molds described above are assembled without the removable wedge for slot formation.

Molding 3l~fl~~d II. Construction of the gel mol(l for preparation of thick-layer gel is illustrated in Fig. 2. By using two frames, each with a

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FIG. 2. 1)esign of gel mold for preparation of thick-layer gel by Method II: (A) shallow dish; (B) cover plate; (C!) gel mold; (D) gel tray; (a,f) glass plates; (b,e) sheets of Saran Wrap; (c,d) plastic frames.

MOLDING METHODS FOR STARCH GEL 237

thickncsa of 3 mm, the shallow tray (Fig. 2A) for filling the gel is made by the same process as above. To deaerate the gel entirely, it is advisable that the heated gel be freed of large air bubbles under negative pressure, transfused into :I sepnratory funnel, and after standing for about 1 min bc drained quickly into the tray. The tray is then, as shown in Fig. 2C, covered immediately with the glass plate covered with Saran, taking care that no bubble is trapped, by lowering the plate obliquely on the tray, with the Saran-covered side downward. The plate is then uniform13 weighted by slight movement of the plate to remove superfluous gel and to obtain an equal thickness of the gel block over the entire surface. After gelation, the mold is turned upside down, the upper glass plate is removed, and the Saran is stripped off the gel surface.

If the two frames illustrated in Fig. 2 are replaced by one frame 1.5 or 2 mm thick, the gel strip, either for the thin-layer or the micro method, can be molded in the same way as above.

Protection of the Gels. After introducing the samples and establishing the bridge connections (5), t.he exposed surface of the gel is sealed with Saran without delay. With the thick-layer gel, the edge of the bridge that was in contact with the gel was introduced into a transverse slot cut in the gel as shown in Fig. 3A or B. When application of the sample either in liquid form or in st’arch grain wicks is made, each surface of the gel

A

f

YIO. 3. “Membrane bridge” set up on surface of thick-layer gel: (a) gel block; (b) gel tray; (c) “membrane bridge”; (d) filter paper layer; (e) transparent plastic membrane ; (f) ndhcsivc vinyl tape.

238 JOJI HORI

divided by a line of sample slots is covered with Saran, and both the slot and the neighborhood are then coated with liquefied petroleum jelly as described by Ramsey (6).

Protein Detection. After electrophoresis the Saran covering (the petroleum jelly seal) and bridge connections are released carefully from the surface of the gel. With the thin-layer technique, after removing the frame from the tray, the gel is transferred easily into a dish for stain- ing and washing by lifting the Saran adhering to the base of the gel. With the thick-layer gel, if desired, the upper frame is removed from the tray and the gel can be sliced horizontally into two layers by means of a cutting blade with the cut along both longitudinal sides of the lower frame of the tray. These layers are stained according to need. The coated Saran can be cut easily with a razor blade if numerous gel segments are desired for eluting protein fractions.

RESULTS AND DISCUSSION

Molding Method I. By using the gel prepared by Method I, starch gel electrophoresis was attempted under a variety of conditions. Considera- tion was given to factors predisposing distortion of the zonal profiles.

First, the influence of irregularity of temperature distribution within the gel on zonal distortion was tested by using either the 6 mm thick layer or the 2 mm thin-layer gel. Application of the sample in liquid form, in starch grain wicks, or in filter paper strips was made. In the horizontal runs, a current density of 6.7 ma/cm2 was applied for 290 min at room temperature, and also for 340 min at 4°C. These conditions generated a sizable amount of heat in the gel. The vertical run was continued under a current density of 3.5 ma/cm2 for 17 hr at room temperature or for 21 hr at 4”, when no temperature rise within the gel was observed. The vertical run was also conducted with cooling of only one side of the gel by a water-cooled plate placed in contact with the bottom of the gel tray. A current density of 9.5 ma/cm2 was applied for 230 min at room tempera- ture during which the temperature of the gel was maintained at 10 + 2°C.

The zonal profile seen in patterns obtained by the above was, as illustrated in Fig. 4A as an example, found in all cases to be vertical to the gel surface; that is, the distortion resulting from differences in thickness of the gel, heat production of the gel during the run, and subse- quent cooling condition could not be recognized. The above suggest that under the above conditions the findings do not coincide with the existing general view (though only one minor factor) that “the main cause for distortion to occur probably lies in the temperature gradient across the thickness of the gel during the run” (1,7,8).

Second, the causes for distortion of the zonal profiles were examined

FIG. 4. Changes occurring in vertical zonal profile on protection of upper side and/or base side of gel prepared by Method I with variety of substances. Vertical runs were carried out without cooling of gel at room temperature by using thick-layer (6 mm) gel. Continuous buffer system of Smithies (4) (borate buffer) was used. Sample of human serum, haptoglobin type 2-2, was put directly into slot in gel. Gel was stained with amido black 1OB. Upper and lower lines of each profile indicate up side and down side of gel, respectively. (A) Saran/gel/Saran; (B) glass/gel/glass; (C) paraffin/gel/glass; (D) paraffin/gel/Saran; (E) Saran/gel/glass; (F) glass/gel/ Saran. Small letters represent zones, and are the same as those in assignments of Smithies (13) : (a) albumin; (b) acidic cY1-glycoprotein; (i) ceruloplasmin; (k,l,m,n) haptoglobin polymers; (0) macroglobulin; (s) transferrin; (t) &lipoprotein; (11) y-globulin. Oblique lines at zonal profile indicate weaker staining.

preliminarily. In this molding procedure, when previous excision of the surface layer of the gel was not made before electrophoresis, vertical profiles of all the zones were clearly distorted (a decrease of electro- phoretic mobility in the upper layer of the gel). At this stage, unnecessary delay in covering the warm gel poured into the dish brought about more marked distortion.

From these experimental results the following hypothesis can be drawn: when the gel solution is poured into a dish placed horizontally the surface tension of the solution increases with the rapid fall in temperature, and gelation probably proceeds rapidly from the surface of the solution with evaporation of water. As a result the concentration along the surface and adjacent layers increases still more, leading thereby to greater intricacy of the network of the gel layer of the surface and adjacent areas when

240 JOJI HORI

compared with the state in t,he deeper layers. The above are believed to be mainly responsible for t,he distortion when the run is conducted under the above conditions. It is known t,hat increase in concent)ration of starch (9) or polyacrylamide gel (10-12) ) though even slight, causes decrease of electrophoretic mobility.

Next, it was ascertained that each side of the gel should be protected with Saran during the run for minimizing the distortion of zones. In the vertical runs, when both sides of the gel were protected with Saran, as shown in Fig. 4A, distortion was practically not observed. Contrarily, all the zones indicated a slight increase of migration in the middle layer of the gel when both sides of the gel were covered with a glass plate (Fig. 4B). Moreover, when both sides of the gel were coated with different substances, the vertical profile of almost all the zones was faintly dis- torted (Fig. 4C and D). The effect of the substance for protecting the gel on distortion has been reascert’ained by reversing the position of different substances in contact with the respective sides of the gel. When the upper and lower sides of the gel were protected with Saran and a glass plate, respectively, as seen in Fig. 4E, many zones exhibited increased migration velocity and weaker staining in the lower layer in contact with the glass. On the other hand, a similar tendency was observed in the upper layer in contact with the glass when Saran and the glass plate covering both sides of the gel were interchanged in position (Fig. 4F).

The phenomena noted may be caused by specific changes resulting in pH, conductivity, field strength, and electroendosmotic flow of both sides of the gel and adjacent layers due to either a physical interaction occur- ring between the gel or buffer and the coating substance (e.g., the poten- tial difference between buffer and coating substance) or adsorption of protein constituents to the coating substance.

One of the main advant.ages of Method I is that the distortion of zones does not practically occur even in the original thick-layer gel, and the other that no visual alteration of staining degree in the zonal profile can be found.2 This method, therefore, is not only recommended for recovery of protein fractions from the gel but also will assure reliability and reproducibility of the result of qualitative observations of patterns on the sliced surface of the gel.2

Molding Method II. By using the gel prepared by Method II, the same tests as above were repeated.” When thick-layer gel was used, as seen in

‘In the horizontal runs, when application of the sample in liquid form was made, a visual alteration of staining degree in eonal profile due to the electrodecantation effect was present.

‘IC was reascertained that differences in heat production of the gel during the run were not the main cause of the distortion.

MOLDING METHODS FOR STARCH GEL 241

Fig. 5A, not a few zones indicated a slight increase of migration in the middle layer of the gel, but these increases were not observed when the thin layer was used. Next, it was checked again that coating both sides of the gel with Saran during the run was necessary to prevent the distortion.

Two precautions must be taken in the actual molding procedure: (1) The mold filled with warm gel should be covered immediately or, after turning over the mold, mobility of components tends to decrease in the base layer of the gel, as shown in Fig. 5B. This suggests again that the

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c -IzIxdklI It RkKKC I w FIG. 5. Changes occurring in vertical zontll plolilc on modification of Method II:

(A) gel prepared by normal procedure; (B) gel prepared by retardation of coating gel after pouring warm gel into tray; (C) gel pepared by turning over mold before gelation. Upper and lower lines of each profile indicate up side and down side of gel after turning over mold, respectively. Thickness of gel, sample, and running condi- tions are the same as thoPe for Fig. 4.

distortion observed in this case will be largely due to the result of increas- ing the gel concentration in the base layer. (2) The mold should be turned over after completion of gelation, or zonal profiles will be bent irregularly (Fig. 5C). Inversion of the mold before gelation might lead to inequality of the gel structure.

In this molding procedure, when inversion of the mold was omitted all the zones obtained indicated a marked decrease of migration velocity in the surface layer (Fig. 6B). Despite the above, the details of why distor- tion was averted by inversion of the mold could not be obtained in the present experiments. Figure 6A’ is an example of the densitometer tracing’ obtained from the thin layer prepared by this molding method in com- parison with the result when inversion of the mold was not made.

As there is no necessity of slicing thtl gel before electrophoresis, the

‘After the gel strip was stained and washed, transmittance densitometry was cam ried out with an Atago self-rerording dcnsitomcter (Tokyo), OZUMOR-6, hy using n filtrr of f110 rn,~.

242 JOJI HORI

B’

FIG. 6. Densitometric tracings (A’,B’) of thin-layer (2 mm) gel patterns with their vertical zonal profiles (A,B). A’ and B’ were obtained from patterns with A and B, respectively. Gel with A was prepared by Method II. Gel with B was prepared with- out inversion of mold in Method II. Sample and running conditions are the same as those for Fig. 4 except for use of filter paper method of sample insertion.

thin-layer gel prepared by Method II has the feature that not only both surfaces of the gel are glassy but also that the thickness of the gel can be maintained uniformly, so that it is suited for scanning the zones in the gel.

These two molding methods also possess the following advantages: (1) each is suitable for either horizontal or vertical starch gel elec- trophoresis, and the samples can be applied in routine methods according to the need; (~7) the struct,ure of the mold is simple, rendering gel molding operation easy, so that these methods are easily standardized for routine use in clinical and research studies; (S) the coating with Saran of both surfaces of the gel prevents the danger of flow of the sample to both surfaces of the gel during the run; (4) the Saran adhering to the base side of the gel facilitates staining and washing of the gel, and removes the

>IOLDING JIETHODS 1cOR STARCH GEL 243

danger of injuring the gel, thus rendering easy the us of gels with comparatively wide surfaces.

SUMMARY

Two improved methods are described for molding starch gel with no distortion in vertical zonal profiles of electrophoresed substrates. Each method is applicable to preparation of the original thick-layer gel as well as of the thin-layer gel by a simple technique, and is useful for qualita- tive and quantitative estimations of apparent distribution of components that penetrate the gel. The results of preliminary considerations of the main causes of distortion of zones are also described.

REFERENCES

1. BAUR, E. W., J. Lab. Clin. Med. 61, 166 (1963). 2. GROULADE, J., AND OLLIVIER, C., Ann. Biol. C&n. 18, 595 (1960). 3. MATSUI, K., AND YAENO, Ii., Anal. Biochem. 6, 491 (1963). 4. SMITHIES, O., Biochem. J. 61,629 (1955). 5. HORI, J., Clin. Chim. Acta 13,642 (1966). 6. RAMSEY, H. A., Anal. Biochem. 5, 83 (1963). 7. BL~EMENDAL, H., J. Chromatog. 3,509 (1960). 8. SOBER, H. A., et al., in “The Proteins” (H. Neurath, ed.), Vol. 3, p. 52, Academic

Press, New York, 1965. 9. SI\IITHIES, O., Arch. Biochem. Biophys., Suppl. 1, 125 (1962).

10. RAYMOND, S., AND NAKAMICHI, M., Anal. Biochem. 3, 23 (1962). 11. RAYMOND, S., AND NAKAMICHI, M., Anal. Biochem. 7, 225 (1962). 12. TOMBS, M. P., Anal. Biochem. 13, 121 (1965). 13. POULIK, M. D., AXD SMITHIES, O., Biochem. J. 68,636 (1958).