Microelectrophoresis on Cellulose Acetate Membranes BENJAMIN W. GRUNBAUM, PAUL 1. KIRK, and WILLIAM A. ATCHLEY Cancer Research Institute and Department o f Medicine, University of California Medico1 Center, and School of Criminology, University of California, Berkeley, Colif.
b Cellulose acetate has been tested as a supporting medium in a new microelectrophoresis apparatus. The method separates serum protein fractions with a speed and clarify far superior to those achieved by conventional filter paper techniques.
I
N A
previous report, Grunbaum and
Kirk (8) described a new type of ap-
paratus for microelectrophoresis which utiliaes a unique device to provide the proper position and tension for t.he filter paper used as the supporting medium. This apparatus i s characterised by its ability to analyze simnltaneously, on the same piece of paper, up
Figure 1. Replicate electrophoretic patterns of same serum sample on strips of varying width
to eight samples ranging from 0.05 to 0.2 pl., and by the rapid resolution of fractions contained in the samples. Recently Kohn (4) descrihed studies of cellulose acetate film as a supporting medium for electrophoresis. This material has been tested in the above a p paratus, and the result of this combination is a technique which appears to be far superior to existing methods of zone electrophoresis. EXPERIMENTAL
Cellulose acetate film (Consolidated Laboratories, Chicago Heights, Ill.) i s first cut into strips 135 mm. long and 29 mm. wide. With the aid of a template and punch seven evenly spaced holes are made, 28 mm. from the end of each strip, t o engage the tensioning device of the apparatus. By removing nzrrow chennels of the acetate film between opposite holes, the film may be divided into any number of strips up to eight (Figure 1). The cellulose acetate membrane i s wetted by allowing it t o drop on to the surface of a tray containing veronal buffer at pH 8.6, 0.075M ionic strength. The membrane is freed of excess buffer by drawing it over the edge of the tray and placed immediately on the apparatus. After serum samples of approximately 0.05 to 0.5 pl. have been placed on the membrane with a pipet or double wire (made by compressing a platinum loop to 3 X 1 mm.), 100 volts are applied for periods ranging from 90 to 120 minutes. A t the end of the desired separation time the membrane i s removed from the apparatus and dried in a forced circulation oven at 100" C. for 5 minntcs. The dye used for staining is PonceauS, 0.2yo in 3% trichloroacetic acid, as recommended by Consden and Kohn ( 2 ) . This stain is preferred over Nigrosine (S), or other conventional protein stains, because it i s possible to clear the membrane of the dye in less than 1 minute. When filter paper is stained with Ponceau-S the background does not wash out in 1hour even after continuous rinsing in 5% acetic acid. The dried membrane is exposed to the dye by floating it onto the surface of the Ponceau solution and, when completely saturated, totally immersing it for 5 to 10 minutes, There is no visible change in staining intensity between 2 minutes and 1 hour. The membrane is then transferred into a,batb of 5% acetic acid and washed until the background is clear, which i s complete after 5 few minutes. Two minutes' drying in an
oven at.
looo
C. adeauatelv nreserves
RESULTS
A number of serum samples were processed in the apparat.us t o test the procedure. Figure 1 shows the results of testing the same sample of serum on 2, 3, 4, and 8 strips simultaneously. In each case the excellent reproducibility of the characteristic pattern, with the advancing boundaries of each sample
Figure 2. Electrophoretic pattern and densitometer study of human serum containing an abnormal homogeneous globulin peak VOL 32, NO. 10, SEPTEMBER 1960
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w t u a l l y on a stla~gllt line, is seen clearly. The strip a t thc bottom shows, in addition to the clear-cut separation of the fractions, the ielative lack of lateral diffusion and absence of tailing. Simple visual euamination of the results obtained with the lien- technique ievealed several differences from patterns obtained with the Spinco Durrum apparatus. With the new apparatus, for instance, a distinct piealbumin band was characteristically present in all serum samples (Figuie 1). The alpha 1 and alpha 2 proteins were more clearly separated, and the relative proportions of all fractions shown by the microelectrophoretic procedure mere somewhat different f i om those shown by the Durrum apparatus. Efforts have becn initiated to place the analysis of the patteins on a quantitative baais by adapting the Spinco Analytrol for use with the cellulose acetate membranes. By proper masking, one can even analyze each narrow strip without detaching it fioni the original membrane. The tlrnsitometric analysis of a broad strip shon n in Figure 2 reveals that each peak wa5 extraordinarily sharp. The pen did not return to the hase line between pciaks only because the slit width %-a
