A Compact

Apparatus

Focusing:

for

Resolution

Vertical

of Rabbit

Polysaccharide

A compact apparatus focusing is described.

for The

analytical apparatus

Gel

Slab

Isoelectric

Anti-Pneumococcal

Antibodies1

and preparative was extremely

comparatively inexpensive. The apparatus trofocusing patterns of anti-pneumococcal different restricted and partially restricted

vertical versatile.

was useful polysaccharide rabbit responders.

gel slab isoelectric easy to use, and

to compare the elecantibodies from several

Polyacrylamide gel electrophoresis, introduced by Raymond and Wang (1). has become widely used as a technique for separating complex mixtures of proteins. This method is useful because good separation can be achieved in a short time. Wrigley (3) has reviewed the use of polyacrylamide gel electrofocusing for biochemical applications. Slab gel electrophoresis, first introduced by Smithies (3) has the advantage that gel homogeneity allows comparison of a number of samples on a single gel. Vertical slab gel electrophoresis (4) was introduced to prevent sample electrodecantation. Several designs incorporating the advantages of slab electrophoresis have already been published (5-10). During the course of study of rabbit anti-pneumococcal polysaccharide antibodies by isoelectric focusing (IEF) (1 I ,I 7). a system was developed which would enable us to make more accurate comparisons of numerous components in purified antibody preparations. It was necessary to construct a highly integrated system which was versatile, convenient to use, and inexpensive. Such a system is described here, and its application to the resolution of antibodies by gel slab IEF is satisfactorily demonstrated. THE APPARATUS” A photograph of the assembled apparatus and its component dimenis shown in Fig. 1. Perspex was used for the main body of the unit.

sions

’ This Canada

investigation (MT-3001)

and

was supported the Ontario

by grants from Cancer Treatment

the

Medical Research Council and Research Foundation

of (No.

244). ” Present :: Fur-ther

address: written

Department of Anatomy (Histology). details of construction may be obtained

University from the

of Toronto. authors upon

request.

VERTICAL

FIG.

I. Assembled

former. I I! teethI with water jacket. with water jacket widths-compartments

apparatus,

GEL

ready

ISOELECTRIC

for

casting

FOCUSING

the

gel:

(A)

45

Per\pe\

multiple-sample

1

slot

I cm apart: (H) upper g:ah outlet. tapped Nylon screw: CC) compartment I9 k 10.X cm: two sections. 6 and I2 mm width\: (D) compartment 1I and electrode chamber-s. 19 x 10.X cm: two xction\. h and 25 mm I and II are hoth comprised of two section\-the thicker of each

having the water jacket milled into grooves adapted for Eppendorf pipet diameter. take\ Tygon tubing (X-mm l-mm bras rings): (H) water jacket.

it. prior to permanent asembly-gluing; (E) tapered tip\: (F) water jacket port\: ((;I \ealing groove. 7 mm Tygon with ?-mm hrasz ring\ or I?-mm Tygon with milled to depth of I cm: (II locking bolt\ (six in total)

showing brass spacer I-ins inserted: (J) serum cap screw. 5 mm >~ 3 cm): (I() lower electrode chamhel-. cm platinum wire inserted into a l-mm groove: (L) hanana plug: tN) gel stopper put in to block off the end\

rod milled to tit electrode of the electt-ode chamber\.

over milled leveling

lower gas outlet I cm diameter dell-in KICW\

chamhcr:

(0)

delrin

(tapped Nylon with 27 gage I5 (3 in total): (M) screw

stoppct

A number of design features are given below: 1. The size of the electrode buffer reservoirs is minimized and the electrodes are recessed into the walls of the chambers. These integrated electrode chambers allow for the adaptation of the apparatus to flat gel (horizontal) electrofocusing ( I3- I5 1. 7,. Water jackets for uniform cooling are milled into both compartments of the unit. The width of the gel chamber is determined by brass spacer rings, and Tygon tubing recessed into paired U-grooves seals the chamber. The chamber size is. therefore. expandable from analytical to preparative dimensions.

452

YEGER

AND

FREEDMAN

3. Polymerized gel is made to extend partially into the electrode chambers, and thus provides for both rigidity and good conductivity. 4. Specially milled grooves on one face conveniently take micropipet tips (Eppendorf pipet) for sample application. METHOD

The theory and method of IEF in acrylamide gels are extensively covered in reviews by Catsimpoolas (16) and Wellner (17). All letters in parentheses refer to Fig. 1. The unit is assembled for a gel run simply by bolting together the two compartments (C and D) after having inserted the brass spacer rings and locking bolts (I) and sealing tubing (G), and screwing in the gel blocking rods (N). The gel chamber can be left dry or is coated with a 1 : 100 dilution of wetting agent (e.g. Photo-F10 200 solution (Kodak), or Column Coat (Canalco)). The gelling solution is 6% recrystallized acrylamide, 0.3% recrystallized NJ’-methylene bisacrylamide; 0.08% N,N,N’,N’-tetramethylenediamine (TEMED), 0.004% riboflavin, and 1% w/v Ampholines, pH 3.5-10. The gelling solution is degassed prior to use. Improvements in resolution have been noted when pH 3.5-10 Ampholines were supplemented with 10% of pH 5-8 Ampholines. A mixture of 1 : 1 pH 4-6 and pH 5-8 Ampholines has proved useful for specific proteins. Addition of from 5-10% sucrose has also resulted in a more stable pH gradient. Photopolymerization was carried out under fluorescent lamps for 30 min. Sample slots were preformed during the initial photopolymerization by means of a Perspex slot former (A). Teflon may give better results, but the wells formed with the Perspex were adequate. Antibody protein concentrations used ranged from 50 to 350 pg per sample with 150 ,ug being ideal (or 5 ~1 of whole serum was also used). Samples are premixed 1: 1 with 50% sucrose containing 5% pH 5-8 Ampholines. After the samples have been added, the remainder of the gelling solution was carefully added and a second photopolymerization carried out. The gel blocking rods (N) are removed (with the gas outlets opened), and the electrode solutions (anode, 0.2% phosphoric acid: cathode, 0.4% monoethanolamine) added to fill 3/4 of the electrode chambers (with the locking bolts in place, I). The apparatus is maintained at a constant temperature of lS”C, and the power supply is connected. The electrofocusing conditions are 1 mA/slot until 350 V is reached, followed by constant voltage for 3-5 hr and finally 500 V for 30 min to sharpen up the banding. After a run, the gel is removed by a reverse sequence of the above. The pH gradient and the isoelectric points of the focused bands were established by cutting out plugs of gel at 0.5-cm sections, incubating the gel plugs in distilled water for I hr (in stoppered tubes) at room temperature, and measuring the pH of the solutions. The gel is easily slipped into a Pyrex dish and is fixed in 10% aqueous trichloracetic acid (TCA) overnight. The gel is stained with 0.2% Coomassie

VERTICAL

GEL

ISOELECTRIC

453

FOCUSING

brilliant blue R250 in 5% TCA, 5% acetic acid, and 30% ethanol. Destaining was complete in 3-5 hr with an improvised activated charcoal unit. The gel was then stored in the acid solution only, and the results were recorded on 35-mm film using a red filter (25A). RESULTS

AND

DISCUSSION

Rabbits immunized with pneumococcal vaccine have been shown to produce a range of antibody responses from highly restricted to unrestricted ( I8,19). To date, the most accurate method to compare the electrofocusing pattern of these responses is IEF in gels, or preparatively in liquid IEF columns (1 I). The conversion of tube systems to slabs was necessary in order that valid comparisons of different antibody populations could be made. We wished to compare both the electrofocusing patterns of multiple bleedings obtained from individual rabbits and the patterns obtained with individual bleedings from several different rabbits. A number of antibody preparations, obtained by salt precipitation and ion-exchange chromatography of the antisera of rabbits immunized with either type III or type VIII pneumococcal polysaccharide, were subjected to gel IEF in a pH 3.5-10 gradient (Fig. 2). The results indicate a high degree of resolution of the samples. but since

gi-1

2

3

4

5

6

7

8

9

IO

11

Ftc;. 2. Vertical gel slab isoelectric focusing of rabbit anti-pneumococcal polysaccharide antibodies. The antibody fractions were focused in a 6% gel using a pH 3.5-10 Ampholine gradient. Two different concentrations were applied (300 wg (samples l-6) and 150 wg (samples 7-l I)). Sample 6 is a restricted anti-type III pneumococcal polysaccharide antibody preparation. and samples I-5 and 7 to I I are partially restricted anti-type VIII pneumococcal polysaccharide antibody populations. Twenty or more distinct bands can be clearly seen in some of the antibody preparations.

454

YEGER

AND

FREEDMAN

similarities in banding do exist, close relationships relative to common antigenic determinants may be indicated. Linearity of the pH gradient was usually obtained and, as previously mentioned, could be improved with the addition of pH 5-8 Ampholines. In order to enhance the resolution of poorly separated protein bands, a second larger apparatus” (I 9 X 19 cm) was constructed. The smaller apparatus (19 X 10.8 cm) had a potential gel running distance of 8 cm, while the larger apparatus had a potential gel running distance of 16 cm. The authors have also used this apparatus to successfully perform alkaline disc gel electrophoresis in urea on antibody light and heavy polypeptide chains, and heavy and light chain separations by isoelectric focusing in polyacrylamide gels in urea. The authors have successfully achieved results comparable to those of Reisfeld and Small (20) and Hoffman er al. (2 I). The versatility of the apparatus is such that it can be readily adapted to the techniques of (a) two-dimensional separations and (b) radioautography. ACKNOWLEDGMENTS We thank Mr. Jack Friel of the Physics Department, structing both the compact and the larger apparati.

University

of Toronto

for con-

REFERENCES 1. Raymond, S.. and Wang. Y. (1960) Anal. Biochrnz. I, 391-396. 7. Wrigley. C. W. (1971) in Methods of Enzymology (Jakoby. W. B. ed.) Vol. 22. pp. 559-564. Academic Press, New York. 3. Smithies. 0. (1955) Riochenl. J. 61, 619-641. 4. Smithies, 0. (1959) Rioche,?~. J. 71, 585-587. 5. Tichy. H. (1966) And. Biochem. 17, 320-326. 6. Reid, M. S.. and Bieleski, R. L. (1968) Atzal. Bioclu~/u. 22, 374-381. 7. Blattler, D. P. (1969) Ad. Biwhem. 27, 73-76. 8. Roberts, R. M., and Jones, J. S. (I 971) Ancrl. Biochen~. 49. 592-597. 9. Chrambach, A.. Pickett. J., Schlam. M. L., Kapadia, G.. and Holtzman. N. A. (1972) Seprrr.

Sci.

IO. Williams. 1 I. Freedman, Eur.

7, 773-783.

J. A., Brand, J. M., and Bosman. T. (1973) M. H.. Pincus. J. H., Yeger. H., McKenney,

J. In~tnunol.

Am/.

Bioch~m.

J. A.. and Mage,

51,

383-389.

R. G. (1974)

4, 553-560.

17. Mage.

K. G., Pincus. J. H.. Alexander, mud. 4, 5 60-564. 13. Awdeh, Z. L.. Williamson, A. R.. and

C., and Freedman. Askonas,

M. H. (1974)

B. A. ( 1968)

Ntrfrrrr

Eur. .I. Im(Lo&o,l)

219,

66-67.

14. Leaback,

D.

H..

and

Rutter.

A. C. (I 9681 Bioc,henz.

Hivl’/!y.v.

RLJJ.

Cotnmr~t~.

32,

447-453.

15. 16. 17. 18. 19. 20. 21.

Radola. B. J. (I 969) Biochim. Biophy.s. Acttr 194, 335-338. Catsimpoolas, N. (I 970) Srpclr. Sr,i. 5, 513-544. Wellner. D. ( 197 1) Anal. Chen~. 43, ( 10). 59A-65A. Haber. E. ( 1970) Fed. Proc. 29, 66-7 I. Haber, E. (1971) A?~I. N. I’. Acud. Sd. 190, ‘85-304. Reisfeld. R. A.. and Small, P. A., Jr. (1966) .Sci~~~ce 152, 1153-1255. Hoffman, D. R.. Grossberg, A. I.., and Pressman, D. (1972j.I. /u?nur~o/.

108,

18-25.