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Polyacrylamide gel electrophoresis on micro slabs
ASALYTICAL
BIOCHEMISTRY
46,
Polyacrylamide
19-32
Gel
H. RAINER
(1972)
Electrophoresis
MAURER
Max-Planck-Itastitut
fiir
AND
Viruaforschung,
Received
February
on
FRANCESCO D
74
Tiibingen,
Micro
Slabs
A. DATI Germany
24, 1971
Polyacrylamide gel electrophoresis, since 1959, has proved to be a very useful and powerful method for fractionating proteins and nucleic acids. Several modifications and advances of the method have subsequently been reported; for a comprehensive monograph see (1). The gel may be cast in cylindrical form (of circular cross-section), as used in disc electrophoresis (2,3), or in flat form (4) (of rectangular cross-section). Each form has its advantages and drawbacks. Flat gels may be conveniently evaluated by quantitative densitometry and photography, without risk of optical artifacts as in the case of cylindrical gels (5). They can be readily dried and subjected to autoradiography and other contact print methods (e.g., for enzyme detection). An additional advantage comes from the use of two-piece gel molds that can be dismantled and thus allow an easy and quick recovery of the gels. The gels remain unscratched and can be stained without delay, thus reducing sample diffusion. Finally, scaling down the gel dimensions from those now in general use should potentiate the feature of flat gels-it should provide: a higher sensitivity of detection due to the smaller cross-sectional area, the opportunity to apply higher voltage gradients because of improved heat dissipation, shorter running periods, and diminished diffusion due t,o more rapid fixation and st#aining of separated sample components. But scaling down of the technique requires increased manual dexterity and training for proper performance to achieve satisfactory separations. This report describes a technique that combines the advantages of flat and micro gels. The technique can be combined with most modifications developed for polyacrylamide gel electrophoresis, e.g., the use of discontinuous buffer systems (to create conditions for true disc electrophorcsis), of gradient gels, and of methods for enzyme, antigen, and radioactivity detection. Several examples will demonstrate the applicability of the technique. Problems arising during performance will be discussed. 19 @ 1972 by
Academic
Press,
Inc.
20
MAURER
AND
DATI
MATERIALS
All chemicals (reagent grade) were purchased from Serva Feinbiochemica, Heidelberg, Germany. Before use, acrylamide and NJ’methylenebisacrylamide were recrystallized from chloroform and acetone, respectively, according to Loening (6). N,N’-Diallyltartardiamide was prepared according to Anker (7). Antisera were obtained from BehringWerke, Marburg, Germany. 14C-Labeled Escherichia coli proteins (approximately 600 dpm/pl) were kindly supplied by Dr. Klein, Friedrich-Miescher-Laboratorium, Tiibingen, Germany. Uhu Plus (Fischer, Biihl, Germany) was used as a two-component epoxy resin glue. METHODS
(1) Micro
Flat Gel Electrophoresis
The gel mold consists of two microscope slides (borosilicate glass) separated by two glass strips as spacers Fig. lA-B). To prepare such a cell, two glass strips (Fig. lA, No. 2) of 75 X 3 X 0.75 mm are fixed onto the longitudinal edges of a normal microscope slide (75 X 25 X 1 mm, No. 3) by means of a two-component epoxy resin glue: In practice, one microscope slide (75 X 25 X 0.7 mm) is glued onto each longitudinal edge and broken, after hardening of the glue, using a glass cutter.
I-
FIO. 1. Equipment for micro flat gel electrophoresis. Gel cell: (A) Cross-section, (B) top view. (1) Cover slide, (la) cover slide with rectangular holes, (2) glass strips, (3) back slide, (4) Teflon comb. (C) Beaker with gel cells. (6) O-ring, (6) plastic net strip. Electrophmesis apparatus: (D) Cross-section, (E) top view. (7) Spongy rubber, (8) upper electrode vessel, (9) Pt electrode, (10) lower electrode vessel.
POLYACRYLAMIDE
GEL
ELECTROPHORESIS
21
The edges are filed smooth. This back slide is covered by another slide (No. l), and the resulting glass cell is held together by two rubber O-rings. To form wells in the gel for sample application a comb (No. 4, cut from a 0.75 mm thick Teflon sheet) is inserted into the upper cell space. A stack of six such cells is suspended on a plastic net strip (nylon mesh, 0.1 mm), and inserted into an appropriate beaker, which is slowly filled with gel solution, carefully avoiding air bubbles. Following polymerization (usually within 25 min), the cell stack is removed by pulling out t’he plastic net and each cell is cleaned from adherent gel. Removal of the Teflon comb must be done slowly to leave sample wells intact. The wells are filled with sample buffer, which is then underlayered with the sample solution made denser with 30% sucrose. Usually cap gels are put on top of the samples to prevent any loss of sample due to dilution or upward diffusion (see “Discussion”). The composition of the cap gels is that of the spacer gel of Table 2 (gel system C). Finally the cells are inserted into slots cut with a knife into a spongy rubber (Fig. ID and E, No. 7), which fits into an opening drilled into t’he bottom of the upper electrode vessel (No. 8). The electrophoresis apparatus is of ordinary design, as used for disc electrophoresis. In a few cases (e.g., for gradient gels), the samples are applied through rectangular holes that have been cut into the cover slide (No. la). (We are indebted to Dr. P. Schneider, Max-Planck-Institut fiir Stromungsforschung, Gottingen, Germany, for preparing such slides.) Prior to filling the cell with gel solution, Teflon stoppers are inserted through the holes until they contact the opposite slide. To prepare different gel layers in the cell for true disc electrophoresis (2,3), the cell is vertically placed into a closed trough. Rinsing a rapidly polymerizing separating gel solution down the glass strips (No. 2) seals the sides and the bottom of the cell, which is allowed to fill up to 3 mm height with the solution. The solution differs from the separating gel solution only by its catalyst concentration, which is doubled. After polymerization, the different gel layers are applied as usual. Following electrophoresis, the cover slide is removed with a spatula, leaving the gel attached to the other slide. The attached gel may be stained by incubation in a solution of 1% Coomassie Brilliant Blue G 250 in 7% acetic acid for 20 min and destained by several washes with 7% acetic acid. Generally, however, we prefer to detach the gel from t’he slide with a wet spatula and to stain it using an alcoholic Coomassie solution (0.5% in methanol/acetic acid/water 4: 1: 5, v/v). All parts of the equipment (slides, rubber, electrophoresis apparatus, etc.) may be reused following cleaning.
22
MAURER
FIG. 2. Setup for production (gradient mixer), (3) magnetic (6) graduated plastic cylinder strip (nylon mesh 0.1 mm), (9)
AND
DATI
of micro gradient flat gels: (1,2) plastic cylinders bar, (4) three-way stopcock, (5) plastic tubing, (layering device), (7) gel cells, (8) plastic net ring, (10) glass column.
(2) Preparation of Continuous Gradient Gels
Essentially the technique of Margolis and Kenrick (8) was adopted. The gradient mixer consists of two graduated plastic cylinders (30 ml) connected by plastic tubing (Fig. 2)) and a magnetic stirrer. Another plastic cylinder (i.d. 32 mm, height 120 mm) with perforated bottom serves as layering device for five cells. Gradient mixer and layering device are connected by plastic tubings. The cells are filled by allowing the gradient mixture to flow from the bottom of the cylinder to the top of the cells at a rate of 34 ml/min. The cell stack sits on a plastic ring (No. 9) to reduce turbulence of the fluid as it enters the ‘mold. A three-way stopcock permits the admission of 30% sucrose solution from a small column (No. 10) to displace the gradient mixture from the tubing and the lower part of the cylinder. Polymerization proceeds from the upper end of the cells downward and is completed within 20 min. (3) Micro Flat Gel Immunoelectrophoresis
Following electrophoresis, a l-l.5 mm thick layer of agar (1% in 0.15 M NaCl containing 0.02% sodium azide) is cast onto the gel according to the technique of Zwisler and Biel (9,lO) (Fig. 3). A 1 mm wide glass capillary (75 mm long) is placed prior to agar casting onto the slab gel in the center parallel to the longitudinal edges of the
POLYACRTLAMIDE
GEL
1
23
ELECTROI’HORESIS
1
FIG. 3. Setup for micro flat gel immunodiffusion: (1) electrophoresed antigen, (2) polyacrylamide gel, (3) agar gel, (4) glass capillary, (5) gel crll (see Fig. 1).
slide. After gelling and removal of the capillary, a clean trough for antiserum application is formed in this way. The proteins are allowed to diffuse out of the gel slab into the agar gel for 24 hr at 20°C. Then antiserum (about 50 ~1) is added. Precipitates appear within 48 hr at 20”. An additional agar layer (1.5 mm thick) is cast in order to strengthen the agar sufficiently for easy removal of the gel slab under water. Washing the gel for 48 hr at 37” with 0.15 M NaCl removes unprecipitated proteins. The precipitin arcs may be photographed directly or subsequent to staining with Coomassie Brilliant Blue G 250 (0.5% in methanol/acetic acid/water 4: 1:5, v/v) and destaining with solvent.
(4) Drying of Micro Flat Gels The gel is spread onto filter paper (Whatman No. 42), covered with a thin plastic film (Saran Wrap) and placed on the porous polyethylene
10 6
FIG. 4. Drying
device for micro flat gels: (A)
cross-section,
(B)
top view. (1)
Gel. (2) filter paper, (3) transparent plastic film, (4) porous polyethylene grid, (5) rubber cover, (6) metal frame, (7) O-ring, (8) vacuum rubber tubing, (9) screw, (10) metal ring.
MATJRER AND DATI
24
FIG. 5. Accessories for contact autoradiography of micro flat gels: (1) dried gel mounted on (2) microscope slide, (3) personal monitoring film, (4) microscope slide, (5) clamp.
grid of the flat gel drying device of Fig. 4. This arrangement mechanically fixes the gel and prevents it from shrinking. Vacuum is applied for 2-3 hr. (5) Contact Autoradiography
of Micro Flat Gels
The dried gel is mounted on a microscope slide by gluing, with the paper support facing the slide. Two “personal monitoring” films (manufacturer: Agfa-Gevaert AG, Leverkusen, Germany; film with 4 notches)
(Component
TABLE 1 Composition of Stock Solutions quantities are given in grams, except for TEMED, 100 ml aqueous solution.)
in ml for
Solution No. Component, Acrylamide Bisa Tris HCl &SO,
1
36.3 to pH
2
5.98 to pH
0.12
4
36.3
5
0.786
6
1.07
to pH 0.109 0.504
HaBOa Glycine EDTA-Nas TEMED” Sucrose PH
3
0.576 0.093 0.23
0.46
8.9
6.7
7
8
9
10
28.0 10.0 32.0 30.0 0.735 2.5 0.6 1.58 to 100 ml with soln. No. 6
0.48 6.0 8.3
9.0
9.0
a Bis = N,N’-methylenebisacrylamide. * TEMED = N,N,N’,N’-tetramethylethylenediamine.
8.3
POLYACRYLAMIDE
GE%
ELECTROPHORESIS
25
are pressed against the slide by means of another slide and two strong clamps (Fig. 5). The second slide provides uniform pressure. The films are exposed for days or weeks at 4°C developed with Kodak D 19b at 20” for 6 min, watered, fixed, and watered again.
The gels are optically scanned by means of the Joyce-Loebl doublebeam microdensitometer (red filter, gray wedge O-l OD). 14C-Scanning is done with the Berthold thin-layer scanner (manufacturer; Berthold, Wildbad, Germany; principle: windowless gas-flow counter, slit width 1 mm). RESULTS
Several gel systems (Table 2) were adapted to the micro method and modified in order to achieve optimum resolution of human serum proteins. Figure 6 presents examples of patterns obtained after electrophoresis in micro flat gels. Gel system A (Fig. 6A), a 7% separating gel, shows a similar separation pattern in comparison with that of the original disc electrophoresis system of Ornstein (2) and Davis (3) : the upper half of the gel reveals a satisfactory resolution of transferrin, haptoglobins, several p- and y-globulins and a,-macroglobulin. In the lower part of the gel, however, Allen’s (11) gel system D, characterized by its uniform pH but discontinuous buffer composition, appears to give better separations in the postalbumin region (Fig. 6C). Decreasing the acrylamide concentration to 5.5% improves the resolution in the region of the high molecular weight globulins, but stacks prealbumins, albumin, and presumably also some postalbumins to a narrow band (Fig. 6D). It follows from this observation that there hardly exists a single gel system which would be optimal for the resolution of all proteins. It is rather advisable, in developing a particular gel system, to limit the goal in each case to optimum separation of the proteins of preferred interest. The recently introduced new cross-linker N,iV-diallyltartardiamide (DATD) (7) produces gels that are more elastic and extensible than gels cross-linked with N,N’-methylenebisacrylamide (Bis) . We found that the weight ratio of acrylamide/DATD should be lower than that of acrylamide/Bis, i.e., it should range around 15 to provide satisfactory sieving properties of DATD-gels and comparable resolutions. This observation is examplified by two gels showing similar IgM protein patterns: the gel of Fig. 6E contains 5.5% acrylamide and 0.275% DATD; the gel of Fig. 6F 5.5% acrylamide and 0.144% Bis. In the latter gel the samples were concentrated, according to the original disc
y See Table to .411en (11).
Riboflavin, 0.1% aq. Tank buffer aoln.a
x4.
Solutiona Solutiona TEMED, 15% aq. DMAYNf 10% aq. .$mmonium persulfat.e, 1%
stock solut,ion
7% separating geP
A
of stock
1.
No. 3
5.Oml
b Modified Ornstein-Davis e Modified Margolis-Kenrick
3
5.Oml
5.5% separating gelb
B
are given
No.7:19.45ml No.1: 6.26ml 6.67 ml
solutions
No.7:25.Oml No.1: 6.26ml 6.67 ml
No.
(quantities
ml
(2,3) system. (8) system.
0.5 ml
No.8:25ml No.2:12.5ml 10.0
spacer gelc
c
5
E 5.5% separating geld
water
No.
to Ornstein-Davis
5
ml
is added
3.5
No.9:19.45ml No.4:15.Oml
c System according f 3-Dimethylaminoproprionitrile.
No.
3.5 ml
No.9:25.Oml No.4:15.Oml
7% separating gel*
D
TABLE 2 Composition of Gel Systems A-F in milliliters; following mixing, distilled solution, except for gel system F) volume
No.
6
(2,3).
6.0 ml
0.5 ml
No.10:14.8ml No. 6: to 100 ml
Soln. A for gradient gels with 4% acrylamidea
to final
No.
6
ml
according
6.Oml
0.18
No.10:75.7ml No. 6: to 100 ml
Soln. B for gradient gels with 20% acrylamidec
d System
F
of 100 ml gel
% :!
g
F CJ g
I’OLTACRTLAMIDE
GEL
ELECTROPHORESIS
27
Fro. 6. Micro flat gel electrophoresis of adult human serum proteins: (A) Densitometric scan of normal protein pattern following electrophoresis in gel system A (Table 2: 7% separating gel) with gel system C as cap gel. (B) Scan of normal protein pattern in a 420% gradient gel (gel system F). (CD) Normal protein patterns in gel syste.m D containing 7%, and gel system E containing 5.5% acrylamide, respectively. (E) IgM protein pattern in gel system B containing 5.5% a&amide and 0275% N,N’-diallyltartardiamide, instead of N,N’-methylenebisacrylamide. (F) IgM protein pattern in gel system B (5.5% separating gel) following protein stacking in gel system C as spacer gel (an original disc electrophoresis system). (G) Normal protein pattern in a 4-20% gradient gel (gel system F) . Range of protein quantity: 10-20 pg in 2-3 pl solution, electrophoresis at 6&1OOV and 3 mA/gel for 100 min at room temperature. Coomaasie staining.
28
MAURER
AND
DATI
electrophoresis method, prior to separation. This additional manipulation did not significantly change the pattern. Since it is now well recognized that the use of gels with continuous concentration gradients may yield better resolutions (8), we attempted to exploit this remarkable potential of the polyacrylamide gel in our micro flat gel technique. It is clear from Fig. 6B and G that several proteins of the pre- and postalbumin region appear as distinct bands not seen in normal disc gels due to partial overlapping with albumin. a-Naphthyl acetate cleaving esterase from rat intestinal epithelium could be readily assayed (12) by means of the gel staining method (1). We found that resolution is better in the micro flat gels than in standard cylindrial gels of 5 or 0.5 mm i.d. Cylindrical gels, more than flat gels, may produce blurred bands on the gel surface.
human serum proteins follou ring FIG. 7. Immunodiffusion pattern of normal electrophoresis in a micro polyacrylamide flat gel (gel system B) and reaction with rabbit antihuman serum in agar. For further details see “Methods.” FIG. 8. Contact autoradiography of ‘C-labeled E. coli proteins electrophoresed in gel system A at 75 V and 3 mA/gel for 10 min at room temperature. Subsequent to drying exposure of film for 14 days at 4°C. For further details see “Methods.”
POLYACRYLAMIDE
GEL
ELECTROPHORESIS
29
Gels prepared and electrophoresed in Plexiglas molds, in contrast to glass cells, showed blurring and tailing bands, particularly in the case of albumin. This observation confirms previous findings (13-15) and forbids the use of untreated Plexiglas. Immunodiffusion techniques could be efficiently combined with our micro slab method, as exemplified by Fig. 7. Following electrophoresis and drying onto filter paper by means of the flat gel drying device of Fig. 4, 14C-labeled E. coli proteins could be detected by thin-layer scanning and contact autoradiography but not by Coomassie staining (Fig. 8). However, a comparison between scanning and autoradiography clearly demonstrated that t,he letter method yields a much better resolution of the labeled proteins. Proteins quantities needed for micro flat gels were found to range between 0.1 and 20 pg contained in 0.5-5 ~1 solution. The sensitivity to detection of a single protein by Coomassie staining amounted to 50 ng. DISCUSSION The main features of micro flat gels should be evaluated by comparison with other gel forms. Cylindrical gels are easy to cast in nonleaking glass tubes and require relatively simple equipment for performing the electrophoresis. They are, however, not suited for autoradiography without longitudinal slicing and may create problems with optical evaluation. Another serious disadvantage is that only a single sample can be analyzed in one particular gel, thus preventing a direct comparison of the separation patterns. In such gels, proteins may migrate for different distances even though they are simultaneously subjected to apparently identical separation conditions in several gel columns. This is caused by small differences in gel length, diameter, or concentration of the gel columns. Different gel densities at the upper gel region may result from the fact that layering of water may not always be achieved in strictly reproducible fashion. In contrast, flat gels allow a simultaneous analysis of several samples and a direct pattern comparison on a single gel, but need more complicated equipment. The advantages of micro gels can, in principle, be obtained with both gel forms. Admittedly, scaling down of dimensions is simpler with gel cylinders and may well be, for technical reasons, the only method for analysis of picogram quantities. Grossbach (16) and Neuhoff (17) have pioneered in developing suitable micro techniques. In summary, micro flat gels offer the following features: small sample quantities (nanograms), analysis of several samples in a single gel, easy removal of the gel for rapid staining and destaining, convenient optical evaluation, drying autoradiography and other contact print methods,
30
MACRER
AND
DATI
high-voltage gradients, quick electrophoresis, and little pattern diffusion. Compared with macro flat gels (>I mm thick) drying of micro slab gels is quicker, preserves the resolution better, and yields uniform and unwrinkled gel films. Furthermore, contact printing met,hods work more economical with micro flat gels since the portion of sample contained in the surface area is greater in these gels than in macro flat gels. This applies in particular to autoradiography. As the micro flat gels can be dried onto filter paper within 2 hr, they may be readily analyzed by this technique. Other methods for radioactivity assay can be used such as direct scanning by means of a thin-layer scanner, and liquid scintillation counting. Cutting the dried “gel paper” yields fractions that may be directly placed into the scintillation liquid or combusted by a suitable micro combustion method (18) to form “Hz0 or l”CO,, for example. Other authors (19-21) have attempted to cast flat gels on microscope slides. Two lines of approach were followed: gel were polymerized (1) in open or (2) in closed molds. The first method used either a high concentration of catalyst (21) or an atmosphere of CO, (19) to surpass the presumed inhibitory action of air-oxygen on polymerization. High concentrations of catalyst may lead to artifacts of the separation patterns (1) and COZ may change the pH. The second method (20) used glass-Plexiglas molds irreversibly fixed to the apparatus. Yet it appears that the resolution obtained with either method does not match that of our micro flat gels. We prefer closed molds with removable comp0nent.s for reasons given above, and obtain resolutions like those of standard disc and thick flat gel electrophoresis. Moreover, we find vertical electrophoresis superior to horizontal as used by some authors (19,21) : air bubbles and buffer leakage causing loss of contact is thus minimized. Problems arising with the use of the micro flat gels should be mentioned : 1. Possible complications may concern leaking of the cell and the formation of air bubbles. The cell may leak, if the glass strips are not precision, to the back slide, which must plainly glued, at utmost provide a uniform contact between cover slide and the strips. Leakage may also occur if the cover slide is moved while handling the cell. Air bubbles may result from too rapid gel polymerization, particularly at edges, and from too quick filling of the cell with gel solution. 2. The ,thickness of the Teflon comb is critical: the comb must just ,fit into the cell with such a clearance as to touch the slides without even slightly pressing them apart. 3. Application of the samples into the gel wells is another critical
POLYACRYLAMIDE
GEL
ELECTROPHORESIS
31
step of the procedure. We prefer using a fine glass capillary filled with sample solution made dense with 30% sucrose and colored with a tracking dye, as bromphenol blue. The capillary is inserted almost down to the bottom of the well, but should not t,ouch the gel to avoid any cause of leakage. It is imperative to release the sample solut,ion slowly, or air bubbles will form. In most cases, we use a cap gel polymerized on top of the sample solution as a upper gel plug, in accordance with the methods of Allen (11). The cap gel contains the leading ion of the multiphasic buffer system and separates the sample from the trailing ion in the upper electrode vessel. Thus, the moving boundary formed by the leading-trailing ions is maintained above the sample during initial separation in the gel underneath the sample. 4. The gels may tend to break while being handled with a forceps or spatula on the air. If this matters, manipulating should be done in a dish filled with water. This, however, may favor diffusion of bands and must therefore be done quickly. 5. Finally, until now, isoelectric focusing did not yield satisfactory separation patterns in our micro gel slabs despite numerous attempts. The reasons for this are obscure. The potential usefulness of the technique for applications in biochemistry, clinical chemistry, and medicine is obvious, e.g., the enzyme patt’ern of small tissue sections of defined origin (biopsy material) can be analyzed. Thus, there is no need to pool different specimens. Moreover, as the method requires only one-tenth to one-twentieth of the sample quantity used for ordinary disc electrophoresis, even very precious materials may be spent for a quick analysis. SUMMARY
An electrophoretic technique using micro polyacrylamide flat gels is described and its usefulness demonstrated. The gels are vertically cast and electrophoresed in slab form (75 X 18 X 0.75 mm) in closed thin glass cells (cuvets) made from detachable microscope slides. The main features of the method are: requirement of small sample quantities (0.1-20 pg contained in
32
MAURER
AND
DATI
ACKNOWLEDGMENTS We thank Miss G. Pauldrach for most valuable technical assistance, Drs. H. B. Strack and D. P. Bolognesi for helpful criticism, and Prof. H. Friedrich-Freksa for continuing support and counsel. REFERENCES “Disc Electrophoresis and Related Techniques of Polyacryl1. MAURER, H. R., amide Gel Electrophoresis,” 2n rev. ed. in English, Walter de Gruyter, Berlin/New York, 1971. (The 1st German ed. was published by De Gruyter Berlin, 196%) 2. ORNSTEIN, L., Ann. N. I’. Acad. Sci. 121, 321 (1964). 3. DAVIS, B. J., Ann. N. Y. Acad Sci. 121, 404 (1964). 4. RAYMOND, S., Cl&. Chem. 8, 455 (1962). 5. ARONSON, J. N., AND BORRIS, D. P., Anal. Biochem. 18, 27 (1967). 6. L~ENING, U. E., Biochem. J. 102, 251 (1967). 7. ANKER, H. S., FEBS Lett. 7, 293 (1970). 8. MARGOLIS, J., AND KENRICK, K. G., Anal. Biochem. 25, 347 (1968). 9. ZWISLER, O., AND BIEL, H., Beringwerk-Mitteilungen Heft 46, 129 (1966). 10. BIEL, H., AND ZWISLER, 0.. Beringwerk-Mitteilungen Heft 46, 141 (1966). 11. ALLEN, R. C., in Ortec 4269 Electrophoresis System Instruction Manual (1969). 12. We are indebted to Dr. N. De Both for performing the enzyme assay (modified method of MARKER, C. L. AND HUNTER, R. L., J. Histochem. Cytochem. 7, 42 (1959)). 13. AKROYD, P., Anal. Biochem. 19, 399 (1967). 14. STUWESANT, V. W., Nature 214, 405 (1967). 15. BLATTNER, D. P., Anal. Biochem. 27, 77 (1969). 16. GROSSBACH, U., Biochim. Biophvs. Acta 107, 180 (1965). 17. NEUHOFF, V., Arzneim. Forsch. (Drug Res.) 18, 35 (1968). 18. 19. 20. 21.
MAURER, H. R., Hoppe-Seyler’s 2. Physiol. Chem. 349, 115 (1968). ALLRED, R. J., AND KEUTEL, K. J., J. Lab. Clin. Med. 71, 179 (1968). CHALVARDJIAN, A., Clin. Chim. Acta 26, 174 (1969). G~Tz, H., SCHEIFFARTH, F., AND EBERL, M., Z. Klin. Chem. Klin. 8, 306 (1970).
Biochem.
BIOCHEMISTRY
46,
Polyacrylamide
19-32
Gel
H. RAINER
(1972)
Electrophoresis
MAURER
Max-Planck-Itastitut
fiir
AND
Viruaforschung,
Received
February
on
FRANCESCO D
74
Tiibingen,
Micro
Slabs
A. DATI Germany
24, 1971
Polyacrylamide gel electrophoresis, since 1959, has proved to be a very useful and powerful method for fractionating proteins and nucleic acids. Several modifications and advances of the method have subsequently been reported; for a comprehensive monograph see (1). The gel may be cast in cylindrical form (of circular cross-section), as used in disc electrophoresis (2,3), or in flat form (4) (of rectangular cross-section). Each form has its advantages and drawbacks. Flat gels may be conveniently evaluated by quantitative densitometry and photography, without risk of optical artifacts as in the case of cylindrical gels (5). They can be readily dried and subjected to autoradiography and other contact print methods (e.g., for enzyme detection). An additional advantage comes from the use of two-piece gel molds that can be dismantled and thus allow an easy and quick recovery of the gels. The gels remain unscratched and can be stained without delay, thus reducing sample diffusion. Finally, scaling down the gel dimensions from those now in general use should potentiate the feature of flat gels-it should provide: a higher sensitivity of detection due to the smaller cross-sectional area, the opportunity to apply higher voltage gradients because of improved heat dissipation, shorter running periods, and diminished diffusion due t,o more rapid fixation and st#aining of separated sample components. But scaling down of the technique requires increased manual dexterity and training for proper performance to achieve satisfactory separations. This report describes a technique that combines the advantages of flat and micro gels. The technique can be combined with most modifications developed for polyacrylamide gel electrophoresis, e.g., the use of discontinuous buffer systems (to create conditions for true disc electrophorcsis), of gradient gels, and of methods for enzyme, antigen, and radioactivity detection. Several examples will demonstrate the applicability of the technique. Problems arising during performance will be discussed. 19 @ 1972 by
Academic
Press,
Inc.
20
MAURER
AND
DATI
MATERIALS
All chemicals (reagent grade) were purchased from Serva Feinbiochemica, Heidelberg, Germany. Before use, acrylamide and NJ’methylenebisacrylamide were recrystallized from chloroform and acetone, respectively, according to Loening (6). N,N’-Diallyltartardiamide was prepared according to Anker (7). Antisera were obtained from BehringWerke, Marburg, Germany. 14C-Labeled Escherichia coli proteins (approximately 600 dpm/pl) were kindly supplied by Dr. Klein, Friedrich-Miescher-Laboratorium, Tiibingen, Germany. Uhu Plus (Fischer, Biihl, Germany) was used as a two-component epoxy resin glue. METHODS
(1) Micro
Flat Gel Electrophoresis
The gel mold consists of two microscope slides (borosilicate glass) separated by two glass strips as spacers Fig. lA-B). To prepare such a cell, two glass strips (Fig. lA, No. 2) of 75 X 3 X 0.75 mm are fixed onto the longitudinal edges of a normal microscope slide (75 X 25 X 1 mm, No. 3) by means of a two-component epoxy resin glue: In practice, one microscope slide (75 X 25 X 0.7 mm) is glued onto each longitudinal edge and broken, after hardening of the glue, using a glass cutter.
I-
FIO. 1. Equipment for micro flat gel electrophoresis. Gel cell: (A) Cross-section, (B) top view. (1) Cover slide, (la) cover slide with rectangular holes, (2) glass strips, (3) back slide, (4) Teflon comb. (C) Beaker with gel cells. (6) O-ring, (6) plastic net strip. Electrophmesis apparatus: (D) Cross-section, (E) top view. (7) Spongy rubber, (8) upper electrode vessel, (9) Pt electrode, (10) lower electrode vessel.
POLYACRYLAMIDE
GEL
ELECTROPHORESIS
21
The edges are filed smooth. This back slide is covered by another slide (No. l), and the resulting glass cell is held together by two rubber O-rings. To form wells in the gel for sample application a comb (No. 4, cut from a 0.75 mm thick Teflon sheet) is inserted into the upper cell space. A stack of six such cells is suspended on a plastic net strip (nylon mesh, 0.1 mm), and inserted into an appropriate beaker, which is slowly filled with gel solution, carefully avoiding air bubbles. Following polymerization (usually within 25 min), the cell stack is removed by pulling out t’he plastic net and each cell is cleaned from adherent gel. Removal of the Teflon comb must be done slowly to leave sample wells intact. The wells are filled with sample buffer, which is then underlayered with the sample solution made denser with 30% sucrose. Usually cap gels are put on top of the samples to prevent any loss of sample due to dilution or upward diffusion (see “Discussion”). The composition of the cap gels is that of the spacer gel of Table 2 (gel system C). Finally the cells are inserted into slots cut with a knife into a spongy rubber (Fig. ID and E, No. 7), which fits into an opening drilled into t’he bottom of the upper electrode vessel (No. 8). The electrophoresis apparatus is of ordinary design, as used for disc electrophoresis. In a few cases (e.g., for gradient gels), the samples are applied through rectangular holes that have been cut into the cover slide (No. la). (We are indebted to Dr. P. Schneider, Max-Planck-Institut fiir Stromungsforschung, Gottingen, Germany, for preparing such slides.) Prior to filling the cell with gel solution, Teflon stoppers are inserted through the holes until they contact the opposite slide. To prepare different gel layers in the cell for true disc electrophoresis (2,3), the cell is vertically placed into a closed trough. Rinsing a rapidly polymerizing separating gel solution down the glass strips (No. 2) seals the sides and the bottom of the cell, which is allowed to fill up to 3 mm height with the solution. The solution differs from the separating gel solution only by its catalyst concentration, which is doubled. After polymerization, the different gel layers are applied as usual. Following electrophoresis, the cover slide is removed with a spatula, leaving the gel attached to the other slide. The attached gel may be stained by incubation in a solution of 1% Coomassie Brilliant Blue G 250 in 7% acetic acid for 20 min and destained by several washes with 7% acetic acid. Generally, however, we prefer to detach the gel from t’he slide with a wet spatula and to stain it using an alcoholic Coomassie solution (0.5% in methanol/acetic acid/water 4: 1: 5, v/v). All parts of the equipment (slides, rubber, electrophoresis apparatus, etc.) may be reused following cleaning.
22
MAURER
FIG. 2. Setup for production (gradient mixer), (3) magnetic (6) graduated plastic cylinder strip (nylon mesh 0.1 mm), (9)
AND
DATI
of micro gradient flat gels: (1,2) plastic cylinders bar, (4) three-way stopcock, (5) plastic tubing, (layering device), (7) gel cells, (8) plastic net ring, (10) glass column.
(2) Preparation of Continuous Gradient Gels
Essentially the technique of Margolis and Kenrick (8) was adopted. The gradient mixer consists of two graduated plastic cylinders (30 ml) connected by plastic tubing (Fig. 2)) and a magnetic stirrer. Another plastic cylinder (i.d. 32 mm, height 120 mm) with perforated bottom serves as layering device for five cells. Gradient mixer and layering device are connected by plastic tubings. The cells are filled by allowing the gradient mixture to flow from the bottom of the cylinder to the top of the cells at a rate of 34 ml/min. The cell stack sits on a plastic ring (No. 9) to reduce turbulence of the fluid as it enters the ‘mold. A three-way stopcock permits the admission of 30% sucrose solution from a small column (No. 10) to displace the gradient mixture from the tubing and the lower part of the cylinder. Polymerization proceeds from the upper end of the cells downward and is completed within 20 min. (3) Micro Flat Gel Immunoelectrophoresis
Following electrophoresis, a l-l.5 mm thick layer of agar (1% in 0.15 M NaCl containing 0.02% sodium azide) is cast onto the gel according to the technique of Zwisler and Biel (9,lO) (Fig. 3). A 1 mm wide glass capillary (75 mm long) is placed prior to agar casting onto the slab gel in the center parallel to the longitudinal edges of the
POLYACRTLAMIDE
GEL
1
23
ELECTROI’HORESIS
1
FIG. 3. Setup for micro flat gel immunodiffusion: (1) electrophoresed antigen, (2) polyacrylamide gel, (3) agar gel, (4) glass capillary, (5) gel crll (see Fig. 1).
slide. After gelling and removal of the capillary, a clean trough for antiserum application is formed in this way. The proteins are allowed to diffuse out of the gel slab into the agar gel for 24 hr at 20°C. Then antiserum (about 50 ~1) is added. Precipitates appear within 48 hr at 20”. An additional agar layer (1.5 mm thick) is cast in order to strengthen the agar sufficiently for easy removal of the gel slab under water. Washing the gel for 48 hr at 37” with 0.15 M NaCl removes unprecipitated proteins. The precipitin arcs may be photographed directly or subsequent to staining with Coomassie Brilliant Blue G 250 (0.5% in methanol/acetic acid/water 4: 1:5, v/v) and destaining with solvent.
(4) Drying of Micro Flat Gels The gel is spread onto filter paper (Whatman No. 42), covered with a thin plastic film (Saran Wrap) and placed on the porous polyethylene
10 6
FIG. 4. Drying
device for micro flat gels: (A)
cross-section,
(B)
top view. (1)
Gel. (2) filter paper, (3) transparent plastic film, (4) porous polyethylene grid, (5) rubber cover, (6) metal frame, (7) O-ring, (8) vacuum rubber tubing, (9) screw, (10) metal ring.
MATJRER AND DATI
24
FIG. 5. Accessories for contact autoradiography of micro flat gels: (1) dried gel mounted on (2) microscope slide, (3) personal monitoring film, (4) microscope slide, (5) clamp.
grid of the flat gel drying device of Fig. 4. This arrangement mechanically fixes the gel and prevents it from shrinking. Vacuum is applied for 2-3 hr. (5) Contact Autoradiography
of Micro Flat Gels
The dried gel is mounted on a microscope slide by gluing, with the paper support facing the slide. Two “personal monitoring” films (manufacturer: Agfa-Gevaert AG, Leverkusen, Germany; film with 4 notches)
(Component
TABLE 1 Composition of Stock Solutions quantities are given in grams, except for TEMED, 100 ml aqueous solution.)
in ml for
Solution No. Component, Acrylamide Bisa Tris HCl &SO,
1
36.3 to pH
2
5.98 to pH
0.12
4
36.3
5
0.786
6
1.07
to pH 0.109 0.504
HaBOa Glycine EDTA-Nas TEMED” Sucrose PH
3
0.576 0.093 0.23
0.46
8.9
6.7
7
8
9
10
28.0 10.0 32.0 30.0 0.735 2.5 0.6 1.58 to 100 ml with soln. No. 6
0.48 6.0 8.3
9.0
9.0
a Bis = N,N’-methylenebisacrylamide. * TEMED = N,N,N’,N’-tetramethylethylenediamine.
8.3
POLYACRYLAMIDE
GE%
ELECTROPHORESIS
25
are pressed against the slide by means of another slide and two strong clamps (Fig. 5). The second slide provides uniform pressure. The films are exposed for days or weeks at 4°C developed with Kodak D 19b at 20” for 6 min, watered, fixed, and watered again.
The gels are optically scanned by means of the Joyce-Loebl doublebeam microdensitometer (red filter, gray wedge O-l OD). 14C-Scanning is done with the Berthold thin-layer scanner (manufacturer; Berthold, Wildbad, Germany; principle: windowless gas-flow counter, slit width 1 mm). RESULTS
Several gel systems (Table 2) were adapted to the micro method and modified in order to achieve optimum resolution of human serum proteins. Figure 6 presents examples of patterns obtained after electrophoresis in micro flat gels. Gel system A (Fig. 6A), a 7% separating gel, shows a similar separation pattern in comparison with that of the original disc electrophoresis system of Ornstein (2) and Davis (3) : the upper half of the gel reveals a satisfactory resolution of transferrin, haptoglobins, several p- and y-globulins and a,-macroglobulin. In the lower part of the gel, however, Allen’s (11) gel system D, characterized by its uniform pH but discontinuous buffer composition, appears to give better separations in the postalbumin region (Fig. 6C). Decreasing the acrylamide concentration to 5.5% improves the resolution in the region of the high molecular weight globulins, but stacks prealbumins, albumin, and presumably also some postalbumins to a narrow band (Fig. 6D). It follows from this observation that there hardly exists a single gel system which would be optimal for the resolution of all proteins. It is rather advisable, in developing a particular gel system, to limit the goal in each case to optimum separation of the proteins of preferred interest. The recently introduced new cross-linker N,iV-diallyltartardiamide (DATD) (7) produces gels that are more elastic and extensible than gels cross-linked with N,N’-methylenebisacrylamide (Bis) . We found that the weight ratio of acrylamide/DATD should be lower than that of acrylamide/Bis, i.e., it should range around 15 to provide satisfactory sieving properties of DATD-gels and comparable resolutions. This observation is examplified by two gels showing similar IgM protein patterns: the gel of Fig. 6E contains 5.5% acrylamide and 0.275% DATD; the gel of Fig. 6F 5.5% acrylamide and 0.144% Bis. In the latter gel the samples were concentrated, according to the original disc
y See Table to .411en (11).
Riboflavin, 0.1% aq. Tank buffer aoln.a
x4.
Solutiona Solutiona TEMED, 15% aq. DMAYNf 10% aq. .$mmonium persulfat.e, 1%
stock solut,ion
7% separating geP
A
of stock
1.
No. 3
5.Oml
b Modified Ornstein-Davis e Modified Margolis-Kenrick
3
5.Oml
5.5% separating gelb
B
are given
No.7:19.45ml No.1: 6.26ml 6.67 ml
solutions
No.7:25.Oml No.1: 6.26ml 6.67 ml
No.
(quantities
ml
(2,3) system. (8) system.
0.5 ml
No.8:25ml No.2:12.5ml 10.0
spacer gelc
c
5
E 5.5% separating geld
water
No.
to Ornstein-Davis
5
ml
is added
3.5
No.9:19.45ml No.4:15.Oml
c System according f 3-Dimethylaminoproprionitrile.
No.
3.5 ml
No.9:25.Oml No.4:15.Oml
7% separating gel*
D
TABLE 2 Composition of Gel Systems A-F in milliliters; following mixing, distilled solution, except for gel system F) volume
No.
6
(2,3).
6.0 ml
0.5 ml
No.10:14.8ml No. 6: to 100 ml
Soln. A for gradient gels with 4% acrylamidea
to final
No.
6
ml
according
6.Oml
0.18
No.10:75.7ml No. 6: to 100 ml
Soln. B for gradient gels with 20% acrylamidec
d System
F
of 100 ml gel
% :!
g
F CJ g
I’OLTACRTLAMIDE
GEL
ELECTROPHORESIS
27
Fro. 6. Micro flat gel electrophoresis of adult human serum proteins: (A) Densitometric scan of normal protein pattern following electrophoresis in gel system A (Table 2: 7% separating gel) with gel system C as cap gel. (B) Scan of normal protein pattern in a 420% gradient gel (gel system F). (CD) Normal protein patterns in gel syste.m D containing 7%, and gel system E containing 5.5% acrylamide, respectively. (E) IgM protein pattern in gel system B containing 5.5% a&amide and 0275% N,N’-diallyltartardiamide, instead of N,N’-methylenebisacrylamide. (F) IgM protein pattern in gel system B (5.5% separating gel) following protein stacking in gel system C as spacer gel (an original disc electrophoresis system). (G) Normal protein pattern in a 4-20% gradient gel (gel system F) . Range of protein quantity: 10-20 pg in 2-3 pl solution, electrophoresis at 6&1OOV and 3 mA/gel for 100 min at room temperature. Coomaasie staining.
28
MAURER
AND
DATI
electrophoresis method, prior to separation. This additional manipulation did not significantly change the pattern. Since it is now well recognized that the use of gels with continuous concentration gradients may yield better resolutions (8), we attempted to exploit this remarkable potential of the polyacrylamide gel in our micro flat gel technique. It is clear from Fig. 6B and G that several proteins of the pre- and postalbumin region appear as distinct bands not seen in normal disc gels due to partial overlapping with albumin. a-Naphthyl acetate cleaving esterase from rat intestinal epithelium could be readily assayed (12) by means of the gel staining method (1). We found that resolution is better in the micro flat gels than in standard cylindrial gels of 5 or 0.5 mm i.d. Cylindrical gels, more than flat gels, may produce blurred bands on the gel surface.
human serum proteins follou ring FIG. 7. Immunodiffusion pattern of normal electrophoresis in a micro polyacrylamide flat gel (gel system B) and reaction with rabbit antihuman serum in agar. For further details see “Methods.” FIG. 8. Contact autoradiography of ‘C-labeled E. coli proteins electrophoresed in gel system A at 75 V and 3 mA/gel for 10 min at room temperature. Subsequent to drying exposure of film for 14 days at 4°C. For further details see “Methods.”
POLYACRYLAMIDE
GEL
ELECTROPHORESIS
29
Gels prepared and electrophoresed in Plexiglas molds, in contrast to glass cells, showed blurring and tailing bands, particularly in the case of albumin. This observation confirms previous findings (13-15) and forbids the use of untreated Plexiglas. Immunodiffusion techniques could be efficiently combined with our micro slab method, as exemplified by Fig. 7. Following electrophoresis and drying onto filter paper by means of the flat gel drying device of Fig. 4, 14C-labeled E. coli proteins could be detected by thin-layer scanning and contact autoradiography but not by Coomassie staining (Fig. 8). However, a comparison between scanning and autoradiography clearly demonstrated that t,he letter method yields a much better resolution of the labeled proteins. Proteins quantities needed for micro flat gels were found to range between 0.1 and 20 pg contained in 0.5-5 ~1 solution. The sensitivity to detection of a single protein by Coomassie staining amounted to 50 ng. DISCUSSION The main features of micro flat gels should be evaluated by comparison with other gel forms. Cylindrical gels are easy to cast in nonleaking glass tubes and require relatively simple equipment for performing the electrophoresis. They are, however, not suited for autoradiography without longitudinal slicing and may create problems with optical evaluation. Another serious disadvantage is that only a single sample can be analyzed in one particular gel, thus preventing a direct comparison of the separation patterns. In such gels, proteins may migrate for different distances even though they are simultaneously subjected to apparently identical separation conditions in several gel columns. This is caused by small differences in gel length, diameter, or concentration of the gel columns. Different gel densities at the upper gel region may result from the fact that layering of water may not always be achieved in strictly reproducible fashion. In contrast, flat gels allow a simultaneous analysis of several samples and a direct pattern comparison on a single gel, but need more complicated equipment. The advantages of micro gels can, in principle, be obtained with both gel forms. Admittedly, scaling down of dimensions is simpler with gel cylinders and may well be, for technical reasons, the only method for analysis of picogram quantities. Grossbach (16) and Neuhoff (17) have pioneered in developing suitable micro techniques. In summary, micro flat gels offer the following features: small sample quantities (nanograms), analysis of several samples in a single gel, easy removal of the gel for rapid staining and destaining, convenient optical evaluation, drying autoradiography and other contact print methods,
30
MACRER
AND
DATI
high-voltage gradients, quick electrophoresis, and little pattern diffusion. Compared with macro flat gels (>I mm thick) drying of micro slab gels is quicker, preserves the resolution better, and yields uniform and unwrinkled gel films. Furthermore, contact printing met,hods work more economical with micro flat gels since the portion of sample contained in the surface area is greater in these gels than in macro flat gels. This applies in particular to autoradiography. As the micro flat gels can be dried onto filter paper within 2 hr, they may be readily analyzed by this technique. Other methods for radioactivity assay can be used such as direct scanning by means of a thin-layer scanner, and liquid scintillation counting. Cutting the dried “gel paper” yields fractions that may be directly placed into the scintillation liquid or combusted by a suitable micro combustion method (18) to form “Hz0 or l”CO,, for example. Other authors (19-21) have attempted to cast flat gels on microscope slides. Two lines of approach were followed: gel were polymerized (1) in open or (2) in closed molds. The first method used either a high concentration of catalyst (21) or an atmosphere of CO, (19) to surpass the presumed inhibitory action of air-oxygen on polymerization. High concentrations of catalyst may lead to artifacts of the separation patterns (1) and COZ may change the pH. The second method (20) used glass-Plexiglas molds irreversibly fixed to the apparatus. Yet it appears that the resolution obtained with either method does not match that of our micro flat gels. We prefer closed molds with removable comp0nent.s for reasons given above, and obtain resolutions like those of standard disc and thick flat gel electrophoresis. Moreover, we find vertical electrophoresis superior to horizontal as used by some authors (19,21) : air bubbles and buffer leakage causing loss of contact is thus minimized. Problems arising with the use of the micro flat gels should be mentioned : 1. Possible complications may concern leaking of the cell and the formation of air bubbles. The cell may leak, if the glass strips are not precision, to the back slide, which must plainly glued, at utmost provide a uniform contact between cover slide and the strips. Leakage may also occur if the cover slide is moved while handling the cell. Air bubbles may result from too rapid gel polymerization, particularly at edges, and from too quick filling of the cell with gel solution. 2. The ,thickness of the Teflon comb is critical: the comb must just ,fit into the cell with such a clearance as to touch the slides without even slightly pressing them apart. 3. Application of the samples into the gel wells is another critical
POLYACRYLAMIDE
GEL
ELECTROPHORESIS
31
step of the procedure. We prefer using a fine glass capillary filled with sample solution made dense with 30% sucrose and colored with a tracking dye, as bromphenol blue. The capillary is inserted almost down to the bottom of the well, but should not t,ouch the gel to avoid any cause of leakage. It is imperative to release the sample solut,ion slowly, or air bubbles will form. In most cases, we use a cap gel polymerized on top of the sample solution as a upper gel plug, in accordance with the methods of Allen (11). The cap gel contains the leading ion of the multiphasic buffer system and separates the sample from the trailing ion in the upper electrode vessel. Thus, the moving boundary formed by the leading-trailing ions is maintained above the sample during initial separation in the gel underneath the sample. 4. The gels may tend to break while being handled with a forceps or spatula on the air. If this matters, manipulating should be done in a dish filled with water. This, however, may favor diffusion of bands and must therefore be done quickly. 5. Finally, until now, isoelectric focusing did not yield satisfactory separation patterns in our micro gel slabs despite numerous attempts. The reasons for this are obscure. The potential usefulness of the technique for applications in biochemistry, clinical chemistry, and medicine is obvious, e.g., the enzyme patt’ern of small tissue sections of defined origin (biopsy material) can be analyzed. Thus, there is no need to pool different specimens. Moreover, as the method requires only one-tenth to one-twentieth of the sample quantity used for ordinary disc electrophoresis, even very precious materials may be spent for a quick analysis. SUMMARY
An electrophoretic technique using micro polyacrylamide flat gels is described and its usefulness demonstrated. The gels are vertically cast and electrophoresed in slab form (75 X 18 X 0.75 mm) in closed thin glass cells (cuvets) made from detachable microscope slides. The main features of the method are: requirement of small sample quantities (0.1-20 pg contained in
32
MAURER
AND
DATI
ACKNOWLEDGMENTS We thank Miss G. Pauldrach for most valuable technical assistance, Drs. H. B. Strack and D. P. Bolognesi for helpful criticism, and Prof. H. Friedrich-Freksa for continuing support and counsel. REFERENCES “Disc Electrophoresis and Related Techniques of Polyacryl1. MAURER, H. R., amide Gel Electrophoresis,” 2n rev. ed. in English, Walter de Gruyter, Berlin/New York, 1971. (The 1st German ed. was published by De Gruyter Berlin, 196%) 2. ORNSTEIN, L., Ann. N. I’. Acad. Sci. 121, 321 (1964). 3. DAVIS, B. J., Ann. N. Y. Acad Sci. 121, 404 (1964). 4. RAYMOND, S., Cl&. Chem. 8, 455 (1962). 5. ARONSON, J. N., AND BORRIS, D. P., Anal. Biochem. 18, 27 (1967). 6. L~ENING, U. E., Biochem. J. 102, 251 (1967). 7. ANKER, H. S., FEBS Lett. 7, 293 (1970). 8. MARGOLIS, J., AND KENRICK, K. G., Anal. Biochem. 25, 347 (1968). 9. ZWISLER, O., AND BIEL, H., Beringwerk-Mitteilungen Heft 46, 129 (1966). 10. BIEL, H., AND ZWISLER, 0.. Beringwerk-Mitteilungen Heft 46, 141 (1966). 11. ALLEN, R. C., in Ortec 4269 Electrophoresis System Instruction Manual (1969). 12. We are indebted to Dr. N. De Both for performing the enzyme assay (modified method of MARKER, C. L. AND HUNTER, R. L., J. Histochem. Cytochem. 7, 42 (1959)). 13. AKROYD, P., Anal. Biochem. 19, 399 (1967). 14. STUWESANT, V. W., Nature 214, 405 (1967). 15. BLATTNER, D. P., Anal. Biochem. 27, 77 (1969). 16. GROSSBACH, U., Biochim. Biophvs. Acta 107, 180 (1965). 17. NEUHOFF, V., Arzneim. Forsch. (Drug Res.) 18, 35 (1968). 18. 19. 20. 21.
MAURER, H. R., Hoppe-Seyler’s 2. Physiol. Chem. 349, 115 (1968). ALLRED, R. J., AND KEUTEL, K. J., J. Lab. Clin. Med. 71, 179 (1968). CHALVARDJIAN, A., Clin. Chim. Acta 26, 174 (1969). G~Tz, H., SCHEIFFARTH, F., AND EBERL, M., Z. Klin. Chem. Klin. 8, 306 (1970).
Biochem.