It should be noted that the cold finger is under a positive pressure when the working gas is confined. When isobutane is the working gas, the pressure is 31 psig. This apparatus has been extensively tested a t pressures up to 109 psig (liquid propane) without failure. Nonetheless, it is recommended that the cold finger be wrapped with adhesive tape as a safety precaution. Although the mercury leveling device described here has been used only with a McLeod vacuum gauge, it should be generally applicable to any laboratory equipment utilizing mercury displacement, e.g., a Toepler pump or gas buret. Received for review March 19, 1973. Accepted June 1, 1973. The support of the United States Atomic Energy (h-fmlission (Contract AT-(40-1)-3606) is gratefully acknowledged.
Figure 1. Modified mercury reservoir with Teflon stopcock assembly and filling stopcock attached A, Mercury reservoir: B. filling stopcock; C, constriction; D , Teflon stopcock; and € , coldfinger
Core Electrophoresis on Polyacrylamide Gel
Rashid A. Zeineh,l Evelyn H. Mbawa, Beverly J. Fiorella,l and George Dunea Cook County Hospital, 7825 W . Harrison St.. Chicago, 111. 60672
Staining is the simplest and easiest method used to localize and quantitate the bands separated by electrophoresis. Starch and polyacrylamide gels are supporting media with a high resolving power because of their molecular sieving effect ( I , 2). Polyacrylamide gel is firmer and more thermostable than starch. It is transparent, nontoxic, relatively inert chemically, and can be prepared with a large range of pore sizes ( 3 ) . Despite these advantages, disc electrophoresis on polyacrylamide gel has not been widely used, because staining is difficult. The gel must be carefully loosened from the tubes with a water stream, then transferred to a staining dish, moved during the destaining process, and then transferred to a test tube for storage and scanning ( 4 ) . Scanning these loose gels in tubes might distort the relative positions of the separated bands. In this report, we describe a method of core electrophoresis on polyacrylamide in which staining is fast, simple, and free of distortion or breakage.
EXPERIMENTAL Immunocore electrophoresis cell and power supply were obtained from Biomed Instruments, Chicago, Ill. Human albumin Behring lot No. 3708 and human transferrin Pentex lot N o . HM3162 were used. Materials for the gels were obtained from Eastman Kodak Company. A 1 2 X 0.6 (i.d.) cm glass tubing is capped on the lower end and a 13 x 0.4 (d.) cm Plexiglas rod is centered in the tube by inserting it in the center cavity of the cap. A centering piece is placed on the upper end to stabilize the rod (Figure 1). The gels are prepared as directed for disc electrophoresis ( 5 ) . The lumen Present address, Chicago Medical School, Department of Microbiology, 2020 West Ogden St., Chicago, Ill. 60612. ( 1 ) 0. Smithies. BiochemJ.. 61, 629 (1955). (21 J. T . Clarke, Ann. N.Y Acad S o , 121. 382 (1964). ( 3 ) 6. J. Davis, Ann. N . Y . Acad. S c i . . 121, 404 ( 1 9 6 4 ) . ( 4 ) I . Smith, in "Electrophoretic Techniques, Vol. I I-Zone Electrophoresis." I . Smith. E d . , 2nd ed.. Interscience Publishers, New York, N . Y . , 1968, p 3 8 2 . (5) / b i d . . p 371
between the tube and the rod is filled with the separating gel using a Pasteur pipet whose tip is in touch with the inside wall of the tube. The separating gel is layered with 1 ml of water and allowed to polymerize for 30-40 minutes. Water is then decanted and the packing gel is introduced with a Pasteur pipet on top of the separating gel. I t is layered with 0.25 ml of riboflavin and allowed to gel for 30 minutes. The caps are removed and riboflavin is decanted (Figure 1, step 1). The tris/glycine buffer is poured into the lower and upper cell compartment undiluted. The sample containing 40% sucrose is applied and electrophoresed for 1 hr a t 5 mA/tube as shown in Figure 1, step 2. After electrophoresis, the rod is withdrawn and the empty core is filled with fixative-dye solution of 1%Bromophenol Blue ( 6 ) or 0.05% Coomassie Blue ( 7 ) stains (Figure 1, step 3). The free dye is rinsed with 7% acetic acid. Another way of staining is by immersing the tube in dye-fixative solution. Purified albumin and transferrin were applied both to disc and core electrophoresis, using 3-, 6-, and 12-inch tubes and the results were compared.
RESULTS The results of core and disc electrophoresis in 3-inch tubes were similar. Disc electrophoresis was found impractical when tubes longer than 3 inch (0.6-cm i.d.) were used because most gels broke while being removed from the tube for staining. Special attention and careful work was needed to remove the polyacrylamide gels. Staining and destaining took from 6 to 12 hours. Core electrophoresis, on the other hand, was safe and simple, and the process of staining and destaining took less than 3 hours when 3-, 6-, or 12-inch tubes were used. For scanning, core electrophoresis tubes with stained gels gave reproducible patterns with constant spacings between separated peaks. By contrast, the use of free gels from disc electrophoresis gave rise to slight variations in the shape and spacing of the peaks. Core electrophoresis
(6) J. Uriel and J . J. Scheidegger, Buli. SOC. Chim B i o i , 37, 165 (1 955), (7) A . Chrambach, R. A . Reisfeld. M . Wyckoff, and A . Zacchari, Ana/. Bfochem..20, 150 ( 1 9 6 7 ) .
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1 2 , OCTOBER 1973
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n SAMPIi
PLEXGLASS R04
CENTERING PIECE
-
PACKING GEL-
SEPARATING GEL
CENTERING CAP
PlPETTE
-
A
DYE-FIXATWE
Figure 1. Step-by-stepiiluslration of t h e technique of core electrophoresis
Figure teins
2.
Core eiectrophoresis of
‘20 pi o f purified
human pro-
In core electrophoresis, the polyacrylamide gels are easily stained because they are not directly manipulated. The use of a plastic rod eliminates errors arising from handling gels or from swelling and breakage. The rigid walls of the tube prevent the gels from expanding and swelling; deformity and breakage do not occur; and the samples can he compared more accurately. A fast staining technique has a clear advantage over a slow one because it immediately stops the diffusion process and thus preserves the resolution obtained during the separation process. During the staining of disc electrophoresis gels, there is a time lapse of about 20-30 minutes per tube while the gels are rimmed and removed from the tubes; the sharpness of the hands decreases and adjacent hands might coalesce because the diffusion continues. This applies especially when many tubes are processed together. Core electrophoresis shortens the time needed to introduce the dye-fixative from 20-30 minutes to a few seconds per tube. In the staining technique described here, diffusion of the stain OCCUIS from the inside to the outside, while with previous methods the staining was from the outside toward the core. Staining is faster because the introduction of a center rod reduces the distance through which the dye must diffuse from 0.3 cm to 0.1 cm. This increases the speed of staining and destaining ninefold. Core electrophoresis can also be used for selective staining of haptoglobins (81, lipoproteins (9),and isoenzymes (10). Its application to study of enzymes (11) and in immunology (12) has proved successful, and it should greatly facilitate the use of column gel electrophoresis in research and in the clinical laboratory. It is concluded that core electrophoresis is an improved form of disc electrophoresis which can also he extended to isoelectric focusing or column chromatography.
Concentrations lor transferrin were 62.5 mg % ( A ) and 250 rng % (01; for albumin 250 mg Vc IC), 4 g % (Dl.and 8 g % (E). Scale (io millimeters) is shown in F
of purified albumin and transferrin gave patterns as shown in Figure 2. The fast bands are the monomer forms and the rest are polymer or molecular forms of that particular protein.
DISCUSSION
Received for review March 5, 1973. Accepted April 16, 1973.
(8) 0. Smithies. Advan. Protein Chem.. 14. 65 (1959). (9) K. A. Narayan, S . Narayan. and F. A. Kumrnerow, Nature. 205. 246
A useful electrophoresis and staining technique should
(1965). (10) A. F . Goldberg. K. Takakura, and R. L. Rosenthal. Nature. 211. 41
allow a high resolution of the separated hands, resulting in greater accuracy of their quantitation and identification.
(1966). (11) R. A. Zeinehand B.J. Fiorella.Clin. Res.. 19. 555 (1971). (12) R. A. Zeineh. 0. 0arret. L. Niemirowski. and B. Fiareila. Amer. J. Physiol.. 222. 1326 (1972).
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