A self-contained microelectrophoresis apparatus

MICROCHEMICAL

JOURNAL

7, 283-286

A ,Self-Contained

(1963)

Microelectrophoresis BEN JAMIN

Environmental

Physiology

Apparatus

W. GRUNBAUM

Laboratory, Department of Physiology, California, Berkeley, California

University

of

AND CARTER Research

and Development

C. COLLINS

Laboratory, University of Califoha San Francisco, California

Medical

Center,

Received August 5, 1963 INTRODUCTION

A microelectrophoresis apparatus recently described by Grunbaum et al. (1-3) and its subsequent uses in combination with cellulose acetate have proved to be most satisfactory in that they permit the separation and quantitation of serum proteins in a fraction of the time required by any other existing technique. For this reason the apparatus is a most desirable tool for the clinical laboratory as well as for research. In the clinical laboratory electrophoresis is routinely performed by the use of a direct current at either a constant voltage or a constant amperage. In our recent investigations employing cellulose acetate with a high voltage drop, 2CL-40 volts per centimeter, the serum proteins have been separated into 6 or 7 major components in 5-15 minutes over distances of IO-30 mm, the degree of separation depending upon the field strength and the concentration of buffer. Thus it is apparent that for the clinical evaluation of serum proteins a 40-mm length of supporting medium would be adequate. This paper describes a miniature version of a microelectrophoresis apparatus recently described by Grunbaum et al. in this journal (4). APPARATUS

The microelectrophoresis apparatus (Fig. 1) contains a simple power supply operated directly from any 11S-volt AC receptacle. Alternatively, it may be operated in the field by means of a small external battery. Two 283

284

BENJAMIN

W. GRUNBAUM

AND

CARTER

C. COLLINS

commercially available 7.5volt batteries (Burgess XXSO) , together measuring only 2% X 2vq inches, will allow the investigator to make over 1000 separations at a cost of only a few dollars. The power supply package can be removed to permit cleaning of the electrophoresis apparatus by complete immersion, or for operation with other external power supplies, if desired. Voltage regulation is unnecessary since for most applications protein separation is practically unaffected by small changes in the field strength such as may result from normal line voltage fluctuations.

FIG. 1.

Microelectrophoresis

apparatus

with

power supply attached.

An interlocking safety switch is incorporated in the cover of the apparatus: voltage cannot be applied to the sample unless the cover is tightly closed. This arrangement precludes operator shock hazard. Figure 2 illustrates the simplicity of the microelectrophoresis power supply. Either 150 or 250 volts DC is easily supplied by appropriate choice of transformer secondary taps. EXPERIMENTAL

The method employed in this study was similar to that described earlier (2) with the exception that, after electrophoresis the cellulose

SELF-CONTAINED

ELECTROPHORESIS

285

APPARATUS

acetate membranes were stained with 0.2yL Ponceau-S dye solution containing 5% sulfosalicylic acid as a coagulant (4). This eliminated the oven drying, which tends to curl the membranes and distort the protein bands.

FIG. 2.

Diagram

of power supply.

FIG. 3. Electrophoretogram of duplicate samples of normal scanning and integration are for one of the serum samples.

human

serum. The

For the relative quantitation of the electrophoresed proteins the cellulose acetate membranes were made permanently transparent according to the procedure of Grunbaum et al. (4). However, for samples in the range

286

BENJAMIN

W. GRUNBAUM

AND

CARTER

C. COLLINS

of 0.25 yl this step may not be necessary because the background density of the untreated cellulose acetate is not sufficient to interfere with the linearity of the densitometric readings of the protein bands. Figure 3 shows an electrophoretogram of normal human serum which illustrates the reproducibility achieved by the method. The densitometer recording made from the same electrophoretogram is also shown, together with the integrated areas under the curve. The complete procedure employing this microelectrophoresis apparatus, including a 1S-minute electrophoresis and the subsequent staining, washing and quantitation, can be accomplished in 30 minutes. When transparentLed, the procedure may take 1 hour. ACKNOWLEDGMENTS Thanks are extended to Mr. Leo Ester-1 for expert assistance in the construction of the pilot model of the microelectrophoresis appartaus, and to Mr. Albert F. Laude1 for technical assistance. REFERENCES 1. 2. 3.

4.

B. W., AND KIRK, P. L., Design and use of a refined microelectrophoresis unit. Anal. Ckem. 32, 564-566 (1960). GRUNBAUM, B. W., KIRK, P. L., AND ATCHLEY, W. A., Microelectrophoresis on cellulose acetate membranes. Anal. Ckem. 32, 1361-1362 (1960). GRUNBAUM, B. W., FESSEL, W. J., AND PIEL, C. F., Densitometric evaluation of microelectrophoretic protein patterns on cellulose acetate membranes. Anal. Chew. 33, 860-862 (1961). GRUNBAUM, B. W., ZEC, J., AND DURRUM, E. L., Application of an improved microelectrophoresis technique and immunoelectrophoresis of the serum proteins on cellulose acetate. Microckem. J. 7, 41-53 (1963). GRUNBAUM,