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E l ~ c r r o p h o r c i i s 1992. I?. 800-802

J A Ulach a n d K. A . Wagganron

Jeffrey A. Black Kimberly A. Waggarnon

Isolab Inc., Akron, OH

Wheat gliadin: Digital imaging and database construction using a 4-band reference system of agarose isoelectric focusing patterns An isoelectric focusing method using thin-layer agarose gel has been developed for wheat gliadin. Using flat-bed units with a third electrode, up to 72 samples per gel may be analyzed. Advantages over traditional acid polyacrylamide gel electrophoresis methodology include: faster run times, nontoxic media, and greater sample capacity. The method is suitable for fingerprinting or purity testing of wheat varieties. Using digital images captured by a flat-bed scanner, a 4-band reference system using isoelectric points was devised. Software enables separated bands to be assigned plvalues based upon reference tracks. Precision of assigned isoelectric points is shown to be on the order of 0.02 pH units. Captured images may be stored in a computer database and compared to unknown patterns to enable an identification. Parameters for a match with a stored pattern may be adjusted for p l interval required for a match, and number of best matches.

Wheat (Triticutri ,aestiviitn L.) as a major cereal crop has received considerable attention in the development of electrophoretic methods for its characterization. Techniques have utilized starch and, most recently, vertical polyacrylamide gel electrophoresis at acid pH of prolamin (gliadin) extracts [I]. For most wheat varieties, the banding patterns of gliadin components are specific to the variety. Because hundreds of varieties are grown in the world, there has been interest in developing schemes to catalogue varieties based upon their electrophoretic patterns. A number of systems of nomenclature have been proposed 12-51, Most of these have been based upon mobility measurements of the gliadin bands. Some have also taken band intensity into account. All have been cumbersome, with manual data entry and subjective judgements of intensity. Recently, with the availability of low-cost personal computers, attention has been given to the possibility of computerized databases of banding patterns along with automated data entry. Shapirstein and Bushuk [ 6 ] digitized mobility values and used a three-band reference system to normalize band positions with a computer algorithm. Although successful in identifying wheat cultivars, acid polyacrylamide gel electrophoresis (PAGE) has drawbacks. Run times are typically long, up to 6.5 h. Coupled with one- to two-day colloidal staining, a minimum of 2 days are needed to obtain a result. The handling of acrylatnide solutions is not without some risk. Sample throughput is limited to about 30 samples per gel. The present report describes an alternative method for gliadin electrophoresis, namely isoelectric focusing (IEF) in agarose gel. Agarose is an ideal medium for IEF: it is nontoxic. Catalysts, which can interact with proteins, are not needed. Proteins, u p to several million molecular weight migrate unrestrkted. With the availability of flat-bed units with a three-electrode design, 70-90 samples may be analyzed on a single gel. Due to the unrestricted migration in this medium, run times even for 200-240 m m gels are 90 min or less. In many cases I E F produces higher resolution

Correspondence: Jeff'rcy A . Black, Isolab Inc. Drawer 4350, Akron, OH 34321, USA

Abbreviations: IEF, ihoelectric focusing; PAGE, polyacrylamide gel electrophoresis: TCA, trichloroacetic acid

than other electrophoretic techniques. Early problems with water exudation with agarose gels have been solved.Anew computer program is described which allows database construction based upon the isoelectric points of separated bands. Gels were obtained from Isolab Inc. (Akron, OH). The dimensions were 203 X 240 X 1 m m containing 2.5% wlv Resolve carrier ampholytes, and 30 O/o ethylene glycol. Varieties of certified spring, winter, and durum wheat were from numerous certification agencies within the United States. All other chemicals used in the study were from Sigma (St. Louis, MO). Single wheat kernels were crushed with pliers between a fold of filter paper. Resultant flour was extracted in a microcentrifuge tube with 60 pL per seed of ethylene glycol [7]. Extractions were carried out a minimum o f 2 h up to overnight with occasional vortexing. After extraction, samples could be used without centrifugation. I E F was performed on either the Multiphor 11,Pharmacia LKB (Piscataway, NJ) or the Desaphor HF, Desaga (Heidelberg) using three electrodes, with the cathode in the center. Circulating coolant was maintained at 15 "C. Interwick distances were 8 cm in all runs. The anolyte was 0.5 M acetic acid, catholyte was 0.5 M sodium hydroxide. Single seed samples (8 pL) were applied 1 cm from the anode with the aid of a polyester sample mask (Isolab). A variety (Minnpro) was arbitrarily chosen as a standard for subsequent scanning experiments. On all runs it was placed at the middle and end positions o n the gel. Gels were run at 30 W constant power for 30 min. At this point the sample mask is removed and the run continued at 60 W for an additional hour. Final voltages were between 1600-1800 V. Gels were fixed in 2OVo wlv trichloroacetic acid (TCA) for 20 min, and washed in 0.5 O/o wlv (TCA) for2 h. Gels were then press-blotted with Schleicherand Schuell GB003 blotting paper and dried in an oven at 70°C. Staining was performed in a solution of 0.1% w/v Brilliant Blue G-250 (diluted from a 1.0% stock in water), 10% w/v phosphoric acid, and 1.0% w/v TCA. Gels are stained within 2 h or may be left overnight. Generally, no destaining is needed. Precipitated surface stain may be removed with a solution of 30% v/v ethanol, 8% v/v acetic acid. Stained gels are stable indefinitely. In separate runs the standard (Minnpro) was run by the described procedure adjacent to p l markers, Pharmacia LKB (Piscataway, NJ). Separated gliadin bands of the standard were assigned 01 73-0838/92/0910-0800 $3.50+.25/0

Ekctropiioresrr 1992, 13. 800-802

Digital iiiiaging of gliadin-patterns by isoelectric focusing i n agarose gel

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Figure I. One half of a gel with separated wheat gliadin used for scanning.The Minnpro standard is at the two end and middle positions.The bands used as references are marked. For the 2-band reference calculations, the bottom and upper bands were used. For the 4-band reference calculations, all four bands were used. Except for the three standard positions, all other lanes contain a different variety. The anode is uppermost.

p l values based upon the calibration curve generated with

the markers. Dried gels were scanned with an Epson ES-300 C color scanner interfaced to an Everex 386/25 computer with 160 MB hard drive and 8 MB RAM. Gel images were displayed on an Everex Evervision color monitor through a Paradise VGA Professional Card. Software was written by Oberlin Scientific (Oberlin, OH) in menu-driven modular form. A complete image of the gel is obtained within 1-2 min. The image is then under software control. The scanner has an X-Y resolution of 300 dots per inch or 84 pm per pixel.The program assigns each pixel a positional value, and an intensity value from 0-255, corresponding to optical density values from 0-2.4. Lanes to be analyzed are selected with the aid of a mouse. Peak and background sensitivities may be individually or globally defined through interactive dialog

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F i g u ~ e 2 .Comparison o f a 2-and 4-band reference system with the standard Minnpro. For n = 11 determinations, each band within the standard lane, detected by the scanner, is plotted against the standard deviation of its calculated isoelectric point.

boxes. The program will calculate plvalues for all the bands in selected lanes based upon the assigned values of bands within the standard lanes. Within a standard lane the number of standard values required is a minimum of 2, with no maximum. The resultant fit was linear from point to point, although other nonlinear fits are capable with the program. The system was tested using 2- and 4-band references within the standard. Figure 1 shows a section of a typical gel used for scanning. For the standard Minnpro, 21 bands were used for subsequent p l calculations. The bands selected as references are marked in Fig. 1.The PI'Swere 5.45, 6.15, 7.30, and 8.50. For the 2-reference system, bands at PI'S 5.45 and 8.50 were used.The 4-band reference used all four. Computer-calculated p l values for the remaining bands within the standard lanes were analyzed for precision. Figure 2 shows graphically the standard deviations obtained for both reference systems. Data was obtained for n = 11 standard lanes over 3 runs. The average standard deviation (SD) for the 2-band reference system was 0.032 pH units, with a maximum value of 0.052. The average SD for the 4-band reference was 0.012 pH units with a maximum value of 0.027. lnspection of the curves reveals increasing imprecision with increasing distance from a reference band with a maximum at midpoints between reference bands. Figure 3 is a photo of a full gel containing 72 samples. The precision obtained with the 4-band reference system is better than the theoretical resolving limit of the gel. Under ideal conditions of heat transfer [8] the resolving power of the gel is 0.028 pH unit / 0.5 mm (actual pH range of the gel is 4.5-9). The program has the ability to store patterns with assigned plvalues in a database. In this way unknown patterns may be compared sequentially with all stored patterns. Matches are based upon the number of similar bands present. The user may select the p l interval required for a match. The number of matches may also be selected and are displayed

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J A Ri ick and K A Wdgganion

E l e c i r o n h o r ~ s i1992. ~ 1.). 800-802

Figure 3. A full gel with 72 samples o r wheat gliadin. The cathode is in the middle.

in descending order of percentage bands matched. Forty varieties have been entered into the database at this point. Preliminary tests have correctlyidentified 11 of I1 varieties. The program will also allow data entry in mobility units. Acid PAGE data is being collected on the same varieties to compare to the IEF results.

lyles. The stain solution is colloidal, and coiit;iins TC:\ to prevent elution of the bands during staining.

The agarose method was relatively fast. Excluding sample preparation, a result is ready for scanning within 5 h. The presence of 3O0/;i ethylene glycol in the gel maintains gliadin solubility during the run and controls water movement in the gel. N o water was observed on the gel surface during any of the IEF experiments. Because the extracted gliadins are soluble in water and alcohols, special precautions in gel processing are required. The 0.5% TCA wash is needed to maintain gliadin solubility while removing carrier ampho-

[ l ] Cooke, R. J., Electrophoresis 1984, S,59-72. [2] Jones, R . W., Taylor, N. W. and Senti, F. R., Arch. Biochem. Bloph.vs. 1959, 84,363-376. [3] Autran, J.C. and Bourdet,A.,Ann. Amreiior, Plunr. 1975,25,277-301. [4] Zillman, R. R. and Bushuk, W., Can. J. Plant Scr. 1978,j8,505-515. [5] Jones. B. L.. Lookharl, G. L., Hall, S . B. and Finney, K. F., Cereal Chem. 1982, j C ) , 181-188. [6] Sapirstein. H. D. and Bushuk, W., Cereal Chem. 1985, 6 j , 372-377. [7] Clements. R. L., Cereal Chem. 1987, 65, 150-152. [S] Righetti, P. G. and Belle, M. S . . Electrophoresis 1992. 13, 275-279.

Reccived August 6, 1992

References

Wheat gliadin: digital imaging and database construction using a 4-band reference system of agarose isoelectric focusing patterns.

An isoelectric focusing method using thin-layer agarose gel has been developed for wheat gliadin. Using flat-bed units with a third electrode, up to 7...
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