Journal of Biochemical and Biophysical Methods, 1 (1979) 209--219 © Elsevier/North-Holland Biomedical Press

REACTION

209

ELECTROPHORESIS

FRANCE BESSETTE Dgpartement de Biophysique, Facultd de Mgdecine, Universitd de Sherbrooke, Sherbrooke, Qug., J I H 5N4 Canada (Received 29 January 1979; accepted 1 May 1979)

The differential mobilities of compounds in an electric field are important analytical criteria and we can use them to bring electrophoretically pure components of a mixture to a selective reaction immediately after their appearance and directly on the carrier medium on which they are separated. To this end, the compounds undergoing reaction are brought into positions on the carrier to assure optimal contact between selected fractions, within a predetermined domain of time and distance. The appearance of a product defines their reactivities, and the product's continued migration on the same carrier can provide the first key to its identity as is demonstrated and discussed. The method is called reaction electrophoresis and it will be of particular use in studies with labile components. It is illustrated here with the coupling reaction of the sodium salt of 1,4-naphthol sulfonic acid and tetrazotized o-dianisidine. Key words: reaction electrophoresis; selective interaction; labile fractions; 1,4-naphthol sulfonic acid; tetrazotized o-dianisidine.

INTRODUCTION T w o o r m o%r e c h e m i c a l s d e p o s i t e d o n a c a r r i e r m e d i u m in t h e a p p r o p r i a t e time and space relation can be driven cataphoreticaUy to react with one a n o t h e r . O n a c y l i n d r i c a l geI, f o r i n s t a n c e , t h e r e a c t i v i t y o f t h e c o m p o u n d s is expressed by the appearance of a product band relative to the position of each individual reactant. The formation of the product and its migration are monitored and then defined by the detection methods that apply best to the case studied. This new technique, called 'reaction electrophoresis', has a wide range of possible applications provided the experimental conditions are j u d i c i o u s l y c h o s e n so as t o r e f l e c t d i f f e r e n c e s in t h e r e a c t a n t s ' p o l a r i t i e s a n d electrophoretic mobilities on the carrier substance. Some advantages of the method are immediately apparent: only small quantities are required to obtain a pure product; labile fractions can be brought to react before d e c o m p o s i t i o n o c c u r s ; t h e p r o d u c t is a l r e a d y c h a r a c t e r i z e d o n t h e c a r r i e r b y

Abbreviations: FBB, Fast Blue B, tetrazotized o-dianisidine; NS, 1,4-naphthol sulfonic acid ; BAG, benzoazurine G.

210

the criteria of electrophoresis. The principle of this method is described and discussed in the following; it is illustrated with the reaction of the sodium salt of 1,4-naphthol sulfonic acid and tetrazotized o-dianisidine on a polyacrylamide gel. MATERIALS

AND

METHODS

(a) Reagents The dye Fast Blue B (tetrazotized o-dianisidine, FBB) undergoes a coupling reaction with the sodium salt of 1,4-naphthol sulfonic acid (NSA). The two reagents carry charges of opposite polarity and react to give benzoazurine G (BAG; Fig. I) [I]. FBB was obtained in practical grade from Sigma Chemical Co. (St. Louis, Mo.) in the form of a stabilized ZnCl2 complex salt with a formula weight of 475.5; the NSA sodium salt (F.W. 246.2) was supplied as practical grade by Eastman Kodak Co. (Rochester, N.Y.); BAG (F,W. 758.5) was purchased from ICN, Inc. (Montreal, Qua.).

(b) Gel eIectrophoresis The buffer used to prepare the acrylamide separation gel was a mixture of 0.25 M potassium dihydrogen phosphate and 0.25 M disodium hydrogen phosphate in a 3 : 7 ratio, giving a pH of 7.~; the addition of 0.46 ml TEMED (N,N,N',N'-tetramethylethylenediamine) per i00 ml brought the pH to 7.5. This buffer is of adequate capacity and guarantees a uniform pH over the length of the gel as its pK of 6.8 is less than i unit removed from the pH at which the experiments were performed. The 7.5% acrylamide separation gel was then prepared as follows: I par~ gel buffer; 2 paz~s of an acrylamide solution containing 30 g acrylamide and 0.8 g Bis (N,N'-methylene-bis-acrylamide) per i00 ml solution; i part distilled water; 4 parts of a OH

2

OCH3

4-

C| N

OCH3

N

N

SO~Na ~ N SA

OH

SO~Na~

FB B

OCH 3 I °

OCH~ ~

BAG

Fig. 1. The model reaction.

OH

SO3Na÷

NCI

211 solution containing 0.14 g perammonium sulfate in 100 ml H20. After introduction of the gel solution into the tubes, water was layered on top and the gels were left to polymerize for 30 m i n . By the criterion of protein mobility as a s t a n d a r d [2], this i s a 'medium pore size' gel Sample gels were prepared as above, with either 0 . 3 mg FBB or with 0.05 mg NSA per ml gel solution except that double the concentration of perammonium sulfate (0.28 g/100 ml H20) was used to polymerize the sample gels containing NSA. The sample gels polymerized also in about 30 min. In experiments where the complete reaction was to be studied, the sample gels containing FBB and NSA, respectively, were formed at opposite ends of the eleetrophoresis tubes; 6 or more tubes of t h e same composition were run at the same time, at 10 mA per tube. The migration of all components was easily followed during a run: FBB is a yellow compound; NSA has a blue fluorescence under UV light; the product BAG has a reddish-purple color at pH 7.5. All gel reagents were electrophoresis grade and purchased from Eastman. Quartz electrophoresis tubes with an inside diameter of 6 mm and a length of 7.5 cm were used throughout (ISCO, Lincoln, Nebr.). Eleetrophoresis apparatus: Buehler PolyAnalyst with a constant voltage/constant current power supply (model 3-1155; Buehler Instruments, Fort Lee, N.J.); the bath temperature was 15 ± 0.1°C.

(c) Detection methods Ultraviolet--visible absorption and fluorescence spectra of the reactants and the reaction product in solution were measured with a Zeiss model DMR-21 recording spectrophotometer and, as required, a Zeiss fluorescence attachment. The positions of the reactant bands were determined by scanning the lengths of the gels, in their quartz tubes, at the absorption maxima characteristic of FBB, NSA and BAG, respectively. For this purpose, the sample compartment of a model 139 Hitachi--Perkin Elmer grating spectrophotometer was modified to accept a scanning attachment. In addition to the instrument slits, a variable slit directly in front of the gel tube defined the width of the light beam to 0.3 mm. A Sage Instruments (Cambridge, Mass.) model 341 infusion-withdrawal p u m p provided the constant scanning speeds of 0.5 and 1.08 cm/min. The spectra were recorded continuously and the positions of the bands measured from t h e clearly visible interface between sample and separation gels. RESULTS AND DISCUSSION

I. Diagrammatic description o f the method The reactants are introduced as samples in gel or solution on a cylindrical separation gel according to their polarity and net charge (Fig. 2). If the

212

(a)

compounds

F e

separation gel >

I~ e

F- e

< reaction product AI+ Bi--* P

electro--phoresis

81 compounds B(+)>

B2 Le

(b)

,-e

L_ e

,-e

--

emP,,sp cooU

~-e

~e

/F\C2

Lm

I~e

re

--e~

c~

r-e

e ~

Lo

Fig. 2. A diagrammatic description of the method, (a) The two reactants have a net charge of opposite polarity. Eleetrophoresis is performed with the reactants incorporated in sample gels or solution(s) at opposite ends of the separation gel. (b) The two reactants are of the same polarity. 1st step: the slower reactant is introduced in sample gel or solution on top of separation gel, and run. 2rid step: the tube is reversed, sample solution poured off, sample gel sliced off. 3rd step: the faster reactant is introduced in sample gel or solution and eleetrophoresis continued.

r e a c t a n t s are o f o p p o s i t e p o l a r i t y ( A - , B*), t h e y are applied at o p p o s i t e ends o f the e l e c t r o p h o r e s i s t u b e as s h o w n in Fig. 2a. T h e y will m o v e in t h e electric field and e v e n t u a l l y c o m e i n t o c o n t a c t (e.g. A1 + BI) to f o r m a react i o n p r o d u c t (P). In t h e c o u r s e of e l e e t r o p h o r e t i c m i g r a t i o n , t h e r e a c t a n t s m a y be s e p a r a t e d as s h o w n (A1, A2, B1, B2). In Fig. 2b, t w o r e a c t a n t s (C-, D - ) c a r r y a charge o f t h e s a m e p o l a r i t y b u t t h e i r e l e c t r o p h o r e t i c m o b i l i t i e s are d i f f e r e n t . In such a ease, t h e slower c o m p o u n d (C-) is m a d e t o r u n t h e field first; t h e r e a c t a n t o f higher m o b i l i t y ( D - ) is t h e n a p p l i e d on t h e reversed gel a n d its f r o n t - r u n n i n g b a n d driven to r e a c t w i t h t h e first c o m p o u n d . It is to b e n o t e d t h a t in either case t h e f a s t e r f r a c t i o n , i.e. t h a t i d e n t i f i e d b y s u b s c r i p t 1, wilt c o m e to r e a c t first. In this case also, b a n d s o f b o t h c o m p o u n d s can be r e a c t e d selectively b y t h e c o r r e c t t i m i n g a n d c o n t r o l o f m i g r a t i o n distances. U n d e s i r e d b a n d s are s i m p l y r u n o f f the gel. T h e p r o d u c t can be c h a r a c t e r i z e d b y its e l e e t r o p h o r e t i c m o b i l i t y if t h e experim e n t is c o n t i n u e d . I t is also possible to e s t i m a t e t h e r e a c t i v i t y o f c o m p o n e n t s t h a t have a l r e a d y m i g r a t e d t h r o u g h a n o t h e r b a n d . T h e r e are n u m e r o u s o p t i o n s and possibilities w h i c h all share t h e a d v a n t a g e t h a t r e a g e n t s b r o u g h t into p o s i t i o n s a p p r o p r i a t e f o r c o n t a c t o n a gel can p r o d u c e p u r e fraetiong w i t h specific reactivities in an e l e c t r o p h o r e s i s s y s t e m such as t h e o n e described here. E v e n labile f r a c t i o n s can be b r o u g h t to r e a c t since we cai~ c o n t r o l t h e m to a p p e a r s h o r t l y or even i m m e d i a t e l y b e f o r e c o n t a c t w i t h ia

213

reactant. Once the reaction is completed needed, by elution or by slicing the gel.

the components

are recuperated, if

H. The choice of the reaction The reaction between the tetrazonium salt, FBB, and the naphthol derivative, NSA, was selected to illustrate the method for the following reasons: the reaction is well defined [i]; the reaction proceeds spontaneously at 15°C for specified conditions; both reactants are soluble in water; the reactants carry charges of opposite polarity; the reaction product does not dissociate in an electric field; the reactants and the reaction product can be detected and distinguished qualitatively by their color or fluorescence and they can be identified quantitatively by spectrophotometric methods. This reaction demonstrates clearly the advantages and shortcomings of the method proposed. The points listed above are, by no means, required for the application of the method, nor do they indicate its limitations. Diazo coupling with phenol or naphthol derivatives is known to take place in alkaline solution [3]. The diazonium salt attaches itself to a position of high electron density and coupling occurs therefore with 1,4-substituted naphthols only at position 2, i.e., at the position ortho to the OH group [4]. The reactivity of FBB and NSA in slightly alkaline solution (pH 7.5) was verified by thin-layer chromatography on silica plates at room temperature.

IIl. Spectral characteristics and stability of the reagents Ultraviolet--visible spectra of the compounds in phosphate buffer at pH 7.2 show maxima at 370 z 3 nm and 306 ± 3 nm for FBB; at 545 z 3 nm and 328 ± 3 nm for commercial BAG, the reaction product. With a mixture of FBB and NSA in the same buffer, one observes an absorption maximum at 525 ± ~ nm for the product. The state of purity of the reactants and/or of commercial BAG and a residual absorption of FBB trailing into longer wavelengths can account for this difference. NSA does not absorb in this region; it has a maximum in the ultraviolet at 296 ± 3 nm with a shoulder at 345 nm (n-~ 7r*). Maximum fluorescence emission is at 436 nm with excitation at 345 nm. Excitation at 296 nm also yields fluorescence at 436 nm but with a three-fold lower intensity. Absorbance was found to be linear with concentrations in the range studied, viz., 0.004 0.04 mg/ml, buffered to pH 7.1, for all three compounds. The stability of the NSA sodium salt in solution (pH 7.1) was verified by taking its ultraviolet--visible spectrum at I0 min intervals over a period of 2 h. No change in absorbance was observed. Samples of NSA in buffer solution, in 7.5% acrylamide solution, and in acrylamide gel of the same percentage turned to the typical reddish-purple color nearly immediately when exposed to saturated solutions of FBB. Solution stability of the zinc chloride cor~plex salt of FBB is rated 'good' [5]. Ultraviolet--visible spectra

214 o f FBB dissolved in pH 7.1 buffer (0.05 mg/ml) and filtered showed a variation of 10% in absorbanee units av the 370 nm peak after 60 rain. Trailing a~ longer wavelengths increases as a function of time. In slightly alkaline solution (pH 7--9~, dissociation of the ZnCI~- complex into the simple chloride sal~ proceeds ~o equilibrium with the h y d r o x i d e ion. Complete conversion so the syn- and anti-diazoate forms is slow and takes 2 3 h. In strong atkali solution, the hydroxide is converted ~o the undissociated anti-diazoa~e so that diazo coupling with naphthol derivatives can no longer occur [6,7], FBB in acrylamide gel solution before polymerization, after 30 min polymerization, and also after an additional 40 min of e!ectrophoresis gave always a reddish-purple p r o d u c t when reacted with solutions of NSA.

IV. Electrophoresis (a) The separate reactants Electrophoresis with NSA dissolved in a 30% sucrose solution buffered to pH 7.5, or, in a 7.5% acrylamide gel at pH 7.5 gave identical results. Under the experimental eonditions as stated earlier, two bands are observed; bot h are fluorescent under ultraviolet light but the leading band is wider and it fluoresces mor e intensely. A plot of their migration distances vs: time is given in Fig. 3a. Immersion of the gel into a saturated sotution of FBB produces a reddish-purple coloration of the leading band; no such reaction with the second band is detectable visually. A quantitative identification by p h o t o m e t r i c scanning was n o t a t t e m p t e d at this point since part of the p r o d u c t had already dissolved in t h e surrounding solution and m ore of it would have been lost by rinsing the gels: The slower and less intense second band which does n o t appear to react [-]

[-]

/

'5

2 l

.m E

(1) ~ . ~

1~

/ ~U

(2)

%

[+] '

1'9

'

2'0

'

3'6

'

4'0

r+l 0;..~ 0

rain time of

. . . . . . .

10

20

30

40 rain

electrophoresis

Fig. 3. Electrophoretic migration of the separate reactants. (a) Anodic migration and separation of NSA. (b) Cathodic migration and separation of FBB. FBB (1) disappears after 25 rain of electrophoresis, n = 10.

215

with FBB is probably an NSA isomer. Ortho isomers are likely to migrate more slowly than the para compounds because of their intra-molecular hydrogen bonding between SOy and the phenolic group. The preparation of naphthol sulfonic acid through sulfonation of l m a p h t h o l yields chiefly the 1,4 derivative along with isomeric acids such as 1-naphthol-2-sulfonic acid, in percentages that vary according to the method used. The presence of 2-naphthol during sulfonation will result in the formation of the 2-naphthol1-sulfonic acid isomer [8]. Naphthol and all its sulfonic acid derivatives are fluorescent; they all couple with diazomum salts in alkaline solution except for 2-naphthol-l-sulfonic acid. However, this latter c o m p o u n d reacts with diazonium salts in acid solution, substituting the sulfonic group with the diazonium group [4]. Immersion of the gel after electrophoresis performed at alkaline pH, in a saturated solution of FBB at pH 4 showed the formation of a colored product at the position of the weak fluorescent band. For these reasons, it seems plausible that the second band contains predominantly 2-naphthol-1 ~sulfonic acid. FBB resolves into two distinct yellow bands already 10 min after the start of the experiment under the conditions mentioned above. Migration distances of both bands are plotted vs. time in Fig. 3b. The leading band is markedly less intense than the second one. When the gel is immersed in a saturated solution of NSA both bands react and show the same coloration, the first b a n d reacting, however, with a delay. After 25 min of migration, the first band can no longer be visually observed, very likely because of diffusion. The two bands represent in all probability fractions of FBB with a different net charge for the reasons given in Section III.

(b ) The reaction NSA and FBB were applied as sample gels on the cathodic and the anodic side, respectively, of the separation gel. Each reactant resolved into two bands as was observed when t h e y underwent electrophoresis separately. Progress of the electrophoretic separation and the appearance of a product was monitored at 5-min intervals for a period of 40 min (Fig. 4). The presence of a second reactant appears not to affect the rate of migration before the formation of a product, as can be seen from the superposition of the lines giving the migration velocities of the separate reactants, taken from Fig. 3. According to the migration pattern, the two leading bands should come into contact after 25 rain. No reaction is observed with the leading FBB band, as was expected for the reasons mentioned above. We notice the formation of a product band by its distinctive color when band 1 of NSA and band 2 of FBB come into contact after 30 rain. The appearance and position of the product band is supported also with ultraviolet--visible scans of the gels prior to and immediately after the reaction (Fig. 5a and b). The tubes are scanned at the absorption maxima characteristic for the reactants, i.e., 550 nm for BAG, at which wavelength the trailing absorption from FBB does not interfere; 380 am rather than 370 nm for FBB to assure separation from

216

o

--

m m

--

--

~

35

'40

(-)

lSA 2

E

8E

7

2

E

:BB

~S

20

'25

~3'D

time of electrophoresis [mini

Fig. 4. R e a c t i o n e l e c t r o p h o r e s i s , n = 8: r e p r e s e n t a t i v e e x p e r i m e n t . F B B a n d N S A are o n t h e same gel; c o n d i t i o n s o t h e r w i s e as in Fig. 3. R e a c t i o n b e t w e e n N S A (1) a n d F B B (2) w i t h f o r m a t i o n o f B A G occurs after 30 m i n . T h e s u p e r p o s e d lines are t h e results o f t h e r e a c t a n t s ' separate e l e c t r o p h o r e t i c migrations~ t a k e n f r o m Fig. 3. A t 15 m i n , n o N S A is left o n t h e s a m p l e gel.

NSA absorption; 300 nm for NSA at which wavelength a contribution from FBB is observed; since, however, both other reagents can be identified separately, this overlap cannot cause a misinterpretation. At 35 rain of electrophoresis, i.e., after the reaction has occured, FBB band 2 is no longer observed indicating that this compound must have been practically used up in the preceding coupling reaction. The new product band migrates towards the anode with a velocity distinctly slower than that of NSA. The migration of NSA not consumed in the reaction is slightly slower after having encountered the FBB band and after leaving the product band behind, as can be seen from a comparison with a migration velocity determined for NSA alone (Fig. 4). The fact that unreacted NSA continues to move towards the anode demonstrates that the stoiehiometry of a reaction on a gel could eventually be worked oul. We come to the conclusion that for this particular reaction with this relatively coarse gel, crossed bands and the formation of a product do not interfere to any serious extent so as to change sensibly the rate of migration. In circumstances different from the model situation described here, the influence of the product band as a series resistance inserted into the conduction pathway will have to be considered. Several eleetrophoretic methods are currently in use to study reactivities; they were, however, designed to identify one of the reactants and they have been applied almost exclusively to the study of immune and enzymatic reactions. These methods can be classified into two general categories: (i) reactants are brought onto a separation gel by application of all electric field; (ii) one of the reactants is uniformly distributed on the separation gel before electrophoresis. In the first category, one finds the technique of immunological analysis by disc-gel electrophoresis after Fitschen [9]. The method measures excess

217

T At 15 min:

~-'~\\

FBB,NSA: FBB :

3 0 0 nm 3 8 0 nm

8AQ,

5~o .m

-

........

FBB

o

At 30 rain:

T8

NSA, FBB~

FBB

g

NSA2

~

NSA

i

0

migration distance [cml

Fig. 5. Ultraviolet--visible scanning of the gels at the wavelengths characteristic for the reactants and the product. (a) Before the reaction, after 15 min of electrophoresis. (b) At the reaction, after 30 rain of electrophoresis.

antigen coming onto the separation gel after passage through a sample gel containing antibody. Since the reaction occurs prior to the separation of the reactants into components, this method does n o t present the advantages of that proposed here in which components can react selectively to obtain an electrophoretically pure product. In a second m e t h o d of this category, counter immunoelectrophoresis on agar cylinders [10] or plates [11], antigen and antibody migrate towards each other, with the antibody moving towards the cathode because the conditions are selected so as to obtain electro-endosmosis. Since the antibody moves very slowly, the distance between the reactants has to be short and the antibody cannot be well resolved electrophoretically [12]. In the method presented here, reactants of the same polarity are brought into contact according to their characteristic electrophoretic migration pattern, with the inherent versatility. A second category of methods in which one reactant is distributed evenly on the separation gel before electrophoresis includes rocket and crossed

218

immunoelectrophoresis [!3,14], both on plates; affinity electrophoresis [15], on plates, used mainly for glycoprotein--lectin systems; the substrateinclusion technique [161, discs, for enzymatic reactions, or modifications thereof, e.g. [!71 for steroid binding proteins. In all these methods, the problem lies in the difficulty of preventing the supposedly immobile reactant from moving during the passage of current [18,191. In the substrate inclusion technique, the gel is incubated to start an enzyme--substrate reaction after the enzyme has been fractionated by eleetrophoresis. In the method proposed here, migration of all reactants and products constitutes the basis for a selective interaction and most of the method's advantages derive from this selectivity. In reaction electrophoresis, several parameters can be modified to control the migration of the reactants and therefore the selectivity of the reaction, viz., the applied voltage, the porosity of the gel, gel--substrate interactions~ etc. The time required to adjust the experimental conditions is, however, offset in many situations by the possibility of obtaining a pure product. SIMPLIFIED DESCRIPTION

OF THE METHOD

AND ITS APPLICATIONS

In a series of experiments to d e m o n s t r a t e a new m e t h o d called 'reaction electrophoresis', t w o dyes differing in the characteristics that define their electrophoretic mobilities were driven by an electric field to c o m e into c o n t a c t and react. A p r o d u c t was o b t a i n e d and identified by its spectral characteristics. S o m e of the conditions, difficulties, and options of this m e t h o d are discussed: (i) electrophoretically pure c o m p o n e n t s or fractions can be brought to react selectively; (ii) labile c o m p o n e n t s or fractions can be reacted i m m e d i a t e l y after their appearance; and (iii) an u n k n o w n p r o d u c t can be characterized by its e l e c t r o p h o r e t i c mobility. A n y potential app!ications derive f r o m these advantages. REFERENCES 1 Welcher, F.J. (1948) Organic Analytical Reagents, Vol. 4, pp. 337--338. D. van Nostrand Co., New Y o r k 2 Maurer, H.R. (1971) Disc Electrophoresis and Related Techniques of Polycaryiamide Gel Electrophoresis, pp. 5--7, W. de Gruyter, Berlin 3 Gurr. E. ( t 9 7 1 ) S y n t h e t i c Dyes in Biology, Medicine and Chemistry, p. 745. A c a d e m i c Press, New Y o r k 4 Venkataraman, K. (1952) The Chemistry of S y n t h e t i c Dyes, Vol. I, pp. 414--424. Academic Press, New Y o r k 5 Liltie, R.D. (1969) in H.J. Conn's Biological Stains (Lillie, R.D., ed.), 8th edn. Williams and Wilkins Co., BaltimOre 6 Saunders, K.H. (1949) The A r o m a t i c Diazo C o m p o u n d s , 2rid edn., pp. 1(}0--101. E. Arnold, L o n d o n 7 V e n k a t a r a m a n , K. (1952) The Chemistry of S y n t h e t i c Dyes, Vol. [, pp. 221 228. Academic Press, New Y o r k 8 V e n k a t a r a m a n , K. (1952) The Chemistry of S y n t h e t i c Dyes, Vol. I, pp. 165--168. Academic Press~ New York 9 Fitschen, W. ( 1 9 6 3 ) Biochem. J. 88, 13P 10 Cr0wle, A.J. (1958) J. Lab. Clin. Med. 48, 642--648 1 t Bussard, A. and Huet, J. (1959) Biochim. Biophys. Acta 34, 258 260

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12 13 14 15 16 17

Hierholzer, J.C. and Barme, M. (1974) J. Immunol. 112,987--995 Laurell, C.B. (1966) Anal. Biochem. 15, 45--52 Laurell, C.B. (1965) Anal. Biochem. 10, 358--361 B~bg-Hansen,T.C. (1973) Anal. Biochem. 56,480--488 Boyd, J.B. and Mitchell, H.K. (1965) Anal. Biochem. 13, 28--42 Weddington, S.C., McLean, W.S., Nayfeh, S.N., French, F.S., Hansson, V. and Ritz~n, E.M. (1974) Steroids 24,123--134 18 Johansson, B.J. (1972) Scan. J. Clin. Lab. Invest. 29, 7--19 19 WadstrSm, T. and Smyth, C. (1973) Science Tools 20, 17--21

Reaction electrophoresis.

Journal of Biochemical and Biophysical Methods, 1 (1979) 209--219 © Elsevier/North-Holland Biomedical Press REACTION 209 ELECTROPHORESIS FRANCE BE...
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