Biochemical Genetics, Vol. 19, Nos. 3/4, 1981

A Simple Method for Preservation and Scanning of Starch Gels

K e n - i c h i N u m a c h i I

Received 16 Apr. 1980--Final 13 June 1980

Starch gels have been the most frequently used supporting medium for zone electrophoresis. Starch gels certainly have advantages over acrylamide gels in the simplicity of gel preparation and the ease of slicing the gel into several thin slices, which can be used for staining various enzymes. However, a tendency of starch gels to curl and crack when drying presents a problem for preservation of gels, and their opacity prevents photometric scanning of minor components. Various methods have been described for increasing the trans- parency of gels (Johns, 1961; Vahvaselk/~, 1962) and for making plastified gels (Groulad et al. , 1961; Bauer et al., 1963). A successful method for preservation of starch gel as a laboratory record was reported by de Ligny (1968), but this method requires photoprint drier and has a number of inherent difficulties. In the present paper, a simple method for the prepara-

t ion of a fully transparent, flexible, dry sheet of starch gel is described. Gels are sliced into 1 or 2 mm thick slices with a wire gel cutter, and

stained in the required fashion. Stained and fixed gel slices are kept in 7% acetic acid solution, washed twice with water to remove excess reagents, and kept in 5% glycerol for 15 to 30 min. The slice is placed on a glass plate of an appropriate size, previously covered with a piece of cellophane sheet. The gel slice is then covered with another sheet of cellophane. Both of the cellophane sheets must be sufficiently large to allow a 2 cm margin on all sides of the glass plate and must be soaked in the glycerol solution for 15 to 30 rain before use. Air bubbles between the layers should be removed by squeezing before subsequent covering is made. An excess amount of glycerol solution poured on the gel facilitates the avoidance of air bubbles when the gel is covered. The

' Otsuchi Marine Research Center, Ocean Research Institute, University of Tokyo, Otsuchi, Iwate 028-11, Japan.

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four edges of the double cellophane holding the gel slice are then folded over the back of the glass. A plastic box smaller than the glass plate on a pan is used as a suitable support of the plate in all the above processes. We routinely note date of experiment, buffer system, and enzyme name stained on a small piece of paper, and put it between the cellophane sheets at the anodal side of the gel, as shown in Fig. 1.

The processed plates are placed on sheets of filter paper in succession. The plates are then covered with another sheet of filter paper, and the excess amount of glycerol is removed by pressing with the palm of the hand. The plates may be left to dry at room temperature, or transferred to a circulating oven, previously set 60 to 80°C. In the former case, further covering with the appropriate paper may be recommended to prevent fading of staining by light. After the gel and cellophane have been dried, the sheets are cut with a razor blade, about 1 or 2 cm from the border of dried gel. The gel and its protective cellophane sheet can easily be stripped off the glass.

The dried gel thus obtained is very transparent and flexible, as shown in

750~3~ J-~EA MDH

Fig. 1. The dried transparent starch gel for electrophoretic variation of NAD dependent malate dehydrogenase in Sebastrobus macrochir (Giinter~. All the letters, showing subunit compositions of isozymes and genotypes of each individual, typewritten on the paper, can be clearly seen through the transparent gel, even though a pale background staining occurs uniformly on the gel by nonspecific reduction of tetrazolium salt.

Preservation and Scanning of Starch Gels 235

1

2

3

Fig. 2. Densitometry of NADP dependent isoci- trate dehydrogenase isozymes in the liver of chum salmon, after separation in the starch gel. Two slices, 1 mrn in thickness, from a single gel, were stained for the same time period. One slice was dried by the method described in the text, and used for contact printing (I) and scanning (2). Another slice was scanned after being rendered transparent by soaking in 99% ethyl alcohol for 30 min, followed by a mixture of benzyl alcohol and glycerol (7:3, v/v) for 3 hr by Aoki's method (3). Some shrinkage occurred in the latter slice. Scan- ning was made on Densitomatic 8, Hikari Denso- kuki, at 570 nm.

Fig. 1. A variety of sizes of gel, even 15 to 25 cm gels, can be dried by this method. The gels prepared by hydrolyzed starch tested so far from various

sources gave the same qualities in t ransparency and flexibility. However, some kinds of starch producing gels of high tensile strength, such as Electrostarch (Madison, Wisconsin), may be preferred when handl ing thin

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slices of gels. Patterns of various dehydrogenases, stained by reduction of tetrazolium salts, and hydrogenase, by azocoupling reactions, were preserved with no alteration during the process of drying. Many have been fully preserved for a storage period of 9 years.

The procedure described here is simpler than previously reported gel preservation techniques, and the dried transparent gel slice is easier to handle and store. Scoring of phenotypes on the gel, measuring the length of migration of bands, and comparing the position of bands between gels, are conveniently carried out in the dried transparent gel. Dried gels can be attached to a data sheet, making it easy to relate each gel to its appropriate data. The dried gel can be photographed by placing it on white paper, and also gives a satisfactory contact print whenever necessary. The transparency of dried gels is also suitable for scanning. Figure 2 shows the results of densitometric scanning of the isocitrate dehydrogenase (NADP dependent) isozyme pattern of chum salmon, Oncorhynchus keta. The minor peaks have been recorded sensitively from the dried gel. All gels in our laboratory have been preserved in this manner.

R E F E R E N C E S

Aoki, K. (1966). Starch gel electrophoresis. In Aoki, A., Nakano, E., and Ohi, U. (eds.), Practical Methods of Electrophoresis (in Japanese), Hirokawa Shoten, Tokyo, pp. 108- 109.

Bauer, E.W., and Steilacoom, F. (1963). Thin layer starch gel electrophoresis and plastification method. J. Lab. Clin. Med. 61:166.

Groulad, J., Fine J. M., and Oliver, C. (1961). Electrophoresis in a thin layer of starch gel followed by a plastifing treatment. Nature 191:72.

Johns, E. W. ( 1961 ). Contact prints of starch gel electrophoresis, J. Chromatog. 5:91. de Ligny, W. (1968). A routine method for the preservation of starch gels. Immunogenet. Lett.

5:128. Vahvaselk~i, E. (1962). Method of quantitative starch-gel electrophoresis. Nature 191:72.