Serum Protein Electrophoresis A Comparison of the Use of Thin-layer Agarose Gel and Cellulose Acetate Louis ROSENFELD, PH.D. Department of Pathology, Clinical Chemistry Laboratory, New York University Medical Center, 560 First Avenue, New York, New York 10016 ABSTRACT Rosenfeld, Louis: Serum protein electrophoresis. A comparison of the use of thin-layer agarose gel and cellulose acetate. Am. J. Clin. Pathol. 62: 702-706, 1974. Thin-layer agarose gel is a relatively new support medium for electrophoresis of serum proteins, but has not been critically evaluated or compared with any other medium. In this report, data for replicate and individual specimen analyses are compared with those obtained on cellulose acetate membranes. The data compare very well. Thin-layer agarose gel offers an advantage in the more distinct separation between beta- and gamma. (Key words: Thin-layer agarose gel; Protein electrophoresis.) THE DEVELOPMENT and utilization of zone electrophoresis for serum protein analysis are too well known to require recounting here. The introduction of cellulose acetate 3 as support medium provided a simplified and vastly improved technic which, characterized by both accuracy and speed, has all but replaced paper. Recently, a new support medium consisting of thin-layer agarose gel 1 was introduced. It offers reproducibility and excellent resolution without background staining. The procedure requires a minimum of time and is simple to use. Methods and Materials The Pol-E-Film system marketed by the Pfizer Diagnostics Division in New York City was used. This includes a specially designed cassette cell and power supply. Agarose Gel. The semisolid agarose gel layer is 0.015-inch thick and is fixed to a transparent flexible plastic backing. The Received January 14, 1974, revised May 2, 1974; accepted for publication May 2, 1974. Address reprint requests to Dr. Rosenfeld. 702 gel consists of % agarose, 5% sucrose, and 0.035% disodium ethylenediamine tetraacetate in barbital buffer, ph 8.6, 0.075 M. Sample wells of uniform dimensions are molded into the gel and are ready to be filled. Electrophoresis Cell. The cell cover contains a cassette to hold and automatically position the agarose film in the buffer compartments. Carbon electrodes run the length of the cell to ensure uniform cell voltage. Power Supply. This is a fixed-voltage direct-current source designed exclusively for this cell. It automatically compensates for line voltage fluctuations from 90 to 125 volts while maintaining a constant output of 90 volts. A voltmeter on the power supply indicates the proper voltage. The thin film permits use of a reduced voltage gradient of 10 volts per cm., with negligible heat build-up and no need for cooling to avoid denaturation of protein. Microliter Pipet. Application of serum specimen is made with a modified Hamilton 10-ju.l syringe with Chaney adapter, and is used with a non-wettable disposable tip.
November 1974 AGAROSE GEL PROTEIN ELECTROPHORESIS 703 Buffer. Barbital buffer, 0.075 M, ph 8.6. One hundred milliliters of buffer are placed in each cell compartment. At least two consecutive runs may be made with the same buffer solution without any effect on the analysis. Stain and Fixative. Amido black 10 B, % in 5% acetic acid. The dye solution may be reused for at least five films if stored in a closed dark bottle when not in use. Procedure Separate the agarose gel film from its protective backing just before a run. Avoid delay at this point to prevent dehydration of the thin agarose layer. Transfer /nl. of serum specimen to a well in the film. If a larger sample is electrophoresed, the intensity of the albumin peak may exceed the sensitivity of the densitometer and the limits of the recording paper used during densitometric quantitation. The Pol-E-Film system directs the use of not more than fi\. serum and suggests that smaller samples may be necessary with some densitometers owing to the characteristics of the light source. Electrophorese for 40 minutes at 90 volts. Stain and fix simultaneously for 15 minutes by immersing in the amido black mixture, gel side up. Remove, let drain, and wash for 30 seconds in 5% acetic acid to remove excess stain. Let drain. Dry at 72 C. for about 20 minutes until the gel is uniformly solid. Failure to dry the gel adequately will result in excessive background staining, which will extend the clearing time. Clear the film by washing with three or more 2-minute rinses with 5% acetic acid. Let drain and dry at room temperature. Quantitation. Densitometric tracing was made with a Beckman Microzone Densitometer (R-l 10) with print-out of data on a Beckman Digital Integrator (R-l II). The processed eight-place agarose film was cut horizontally in half to fit inside the clear plastic envelope used for cleared microzone cellulose acetate membranes. A 550- nm. interference filter should be used instead of one at 580 nm. as recommended for the Pol-E-Film system. Cellulose Acetate. The Beckman Microzone electrophoresis cell 2 and procedure were used according to the manufacturer's instructions, 4 except that a shorter migration time (14-16 minutes) was found adequate for separation of the proteins. Staining and fixing were done with an aqueous solution of % Ponceau S containing 3% sulfosalicylic acid and 3% trichloroacetic acid. The membrane was subsequently cleared, and scanned in the computing densitometer with a 520-nm. interference filter. Results Sixty-nine specimens of serum from outpatients and hospitalized individuals in whose cases routine requests for protein electrophoresis had been made were randomly selected for analysis by both technics. Total protein concentrations ranged from 3.9 to 8.7 Gm. per dl., and were determined with biuret reagent on an Auto- Analyzer. Seven of these were selected by visual inspection for apparent differences in the appearances of their patterns, and each was rerun in replicate analyses, eight on a film and membrane. Quantitation was made in terms of percentage of total densitometric area scanned for each of the five protein fractions. A pre-albumin fraction always separated out on the agarose gel and, although visible to the unaided eye when viewed against a white background, it was usually too faint to register more than a slight rise in the baseline of the tracing, and was not included in the calculation. The data for the 69 serum specimens are shown in Table 1, and include mean and standard deviation () for each of the five protein fractions analyzed by each technic. Coefficients of variation ( = /mean) are not stated for these data, because they merely define the range of sera studied. Also included in Table 1 are the linear regression equation by the
704 ROSENFELD A.J.C.P. Vol. 62 Table 1. Electrophoretic Fractionation of 69 Serum Specimens on Agarose Gel (y) and Cellulose Acetate (x) Fraction Agarose Gel Cellulose Acetate Linear Regression Equation Coefficient ofcorrelation 5 7.5 5 8.1 y = 5.56 + 8 x 5 Alpha-1-globu in 5.8 4.0 y = 9+ l.lox 9 Alpha-2-globu lin 1 1 y = 2.15 + 0x 7 Beta- 1 y = 4 + 4 x 0 Gamma-globu in 16.6 2 y = -8 + 2 x 5 Table 2. Electrophoretic Fractionation on Agarose Gel and Cellulose Acetate of Serum Specimens with Values below 45% and above 60% Alpha-1- Alphas Gammaglobt lin * t 45.7 44.7 44.7 4 4 4 4 39.7 34.0 33.4 31.7 27.2 4 48.8 47.2 4 42.2 39.1 42.1 38.2 3 3 31.7 21.7 9.4 5.5 1 4.9 7.2 6.3 3.4 9.5 5.0 2.7 17.9 1 1 1 19.9 9.7 1 7.6 15.7 32.5 16.3 1 19.6 1 1 34.9 1 1 1 19.5 1 1 19.2 19.2 2 12.1 1 1 1 19.4 12.9 1 15.8 12.9 24.9 18.1 2 2 19.1 34.1 2 3 3 25.0 1 2 15.5 25.7 29.1 20.1 18.2 38.2 27.4 39.4 4 33.5 1 39.0 38.9 7.9 1 15.2 15.1 3.9 4.4 23.3 26.8 9.6 6 61.7 6 65.0 67.9 62.1 64.8 64.6 6 69.4 3.3 3.5 2.7 1 1 6.8 8.2 8.9 9.2 13.4 14.8 1 10.1 10.0 6.6 1 1 1 1 6 64.7 4.1 8.5 13.4 1 8.8 1 * Agarose gel. t Cellulose acetate.
November 1974 AGAROSE GEL PROTEIN ELECTROPHORESIS 705 Table 3. Replicate Analyses (n = 8) of Seven Serum Specimens by Electrophoresis on Agarose Gel and Cellulose Acetate Alb umin Alpha-1- Alpl ia-2- Gamma * t Serum 1 6 6 17.4 7.2 7.6 7.9 1 9.9 18.7 4.8 Serum 2 54.4 54.4 4.1 2.2 9.1 9.3 8.2 15.1 13.5 1 4.7 2 1.2 Serum 3 53.3 5 2.5 1 16.3 2.9 15.0 1 1 Serum 4 5 6 25.0 8.0 7.5 1 1 1.2 8.5 17.4 Serum 5 53.4 2.1 5 8.9 1 1 6.3 11.7 14.7 Serum 6 54.7 52.1 8.6 1 4.0 1 1 4.3 1 5.5 17.2 1.7 Serum 7 59.7 1.7 55.8 1 4.9 10.0 5.0 1 1 18.7 * Agarose gel. t Cellulose acetate. Table 4. Average Coefficients > of Variation of Seven Sets of Replicate Data Alpha-l Alphas Gamma Agarose gel Cellulose acetate 1 3.3 3.9 method of least squares and Pearson's coefficient of correlation, r, for each fraction. All statistical calculations were made on an Olivetti Programma 101 with programs supplied by the manufacturer. Inasmuch as these means were derived from a wide range of data, a comparison of two subgroups of the samples was also made. Distinctly abnormal specimens, all those with albumin levels below 45%, are compared individually by both technics in Table 2. Also compared are the results of those sera with albumin values above 60%. The data of the seven sets of replicate
706 ROSENFELD A.J.C.P. Vol. 62 analyses (mean,, C.V. %) appear in Table 3, and the averages of these coefficients of variation in Table 4. According to the spectral-transmittance curve of amido black there is maximum absorption from 610 to 625 nm. The Pol-E- Film system calls for density measurement at 580 nm. In a personal communication, the manufacturer states that any wavelength between 550 and 580 nm. should be suitable, depending on the optical design and characteristics of the densitometer. They recommend that the instrument be calibrated for wavelength with sera of known electrophoretic composition. Widebandpass filters of colored glass with maximum transmission at 570 and 595 nm. were not effective in the densitometer. Interference filters at 520, 550,570, and 600 nm. were tested, and the percentage albumin fractions of 16 samples on agarose gel were compared with values obtained with the cellulose acetate procedure. With the 600 or 570 nm. filter, the values for albumin were 5-10% less than with the cellulose acetate procedure, while at 520 nm. they were 3-5% greater. Results at 550 nm. on agarose gel compared very well with values obtained on cellulose acetate and are presented in this report. Discussion There is good agreement between the two technics for the five component proteins separated by electrophoresis. This is especially evident when comparison is made on an individual basis for specimens covering a wide range of abnormal values (Table 2). It is apparent from Table 3 that reproducibility with thin-layer agarose gel compares well with that obtained with cellulose acetate. When the coefficients of variation for each fraction are averaged (Table 4), it can be seen that the values are consistently low and that the reproducibility with agarose is as good as that with cellulose acetate. In the comparisons for the total group (Table 1) there is little difference between the 's of the methods for each fraction. The means also are very similar, except for beta- and gamma-. Examination of the densitometric tracings shows a more distinct separation between beta and gamma- on agarose gel, and a less homogeneous and less distinct beta configuration on cellulose acetate for some membrane runs. In fact, it is difficult to characterize the occasional extra peak in the vicinity of specimen application on cellulose acetate as beta-2 or gamma-1, and this probably contributes to the discrepancy between these two fractions. This sixth peak appears as a shoulder on the gamma or between gamma and beta. It may, in part, be an artifact resulting from deviations in technic which lengthen the migration distances of the fractions. This may also be due to batch-to-batch differences in membrane substructure which cause variations in degree of hydration and result in a non-uniform electrical field and distorted separation. The higher water content of the agarose gel insures uniformity of the electrical field and produces better definition of the gamma region. The high degree of precision may be explained by the relatively neutral characteristics of agarose, the minimal electroendosmosis, and the optical clarity and porosity. 1 Acknowledgment. Carlito Esquivel provided technical assistance. References 1. Elevitch FR, Aronson SB, Feichtmeir TV, et al: Thin gel electrophoresis in agarose. Am J Clin Pathol 46:692-697, 1966 2. Grunbaum BW, Zee J, Durrum EL: Application of an improved microelectrophoresis technique and Immunoelectrophoresis of the serum proteins on cellulose acetate. Microchem J 7:41 53, 1963 3. Kohn J: Small scale membrane filter electrophoresis and immuno-electrophoresis. Clin Chim Acta 3:450-454, 1958 4. Manual 015-083618-A, Beckman Instruments, Inc., Fullerton, California, August 1972