Isoelectric focusing in immobilized pH gradients: Principle, methodology and some applications

Journal of Biochemical and Biophysical Methods. 6 (1982) 317-339 Elsevier Biomedical Press

317

Isoelectric focusing in immobilized pH gradients" Principle, methodology and some applications * Bengt Bjellqvist ~, Kristina Ek 1, Pier Giorgio Righetti 2, Elisabetta Gianazza 2, Angelika GOrg 3. Reiner Westermeier 3 and Wilhelm Postel 3 / LKB Produkter AB, Box 305, S-161 26 Bromma, Sweden. 2 Department of Biochemistry, Uniw,rsttyof Mdan, Vta Celorta 2, Milan 20133, Italy. and 3 Techmcal University of Munich, D-8050 Freising- Weihenstephan. F.R. G.

(Received 10 June 1982) (Accepted 14 June 1982)

Summaw A new technique for generating pH gradients in isoelectric focusing is described, based on the principle that the buffering groups are covalently linked to the matrix used as anticonvective medium. For the generation of this type of pH gradient in polyacrylamide gels. a set of buffering monomers, called Immobiline (in analogy with Ampholine). is used. The pH gradient gels are cast in the same way as pore gradient gels. but instead of varying the acrylamide content, the light and heavy solutions are adjusted to different pH values with the aid of the lmmobiline buffers. Available immobiline species make it possible to generate any narrow linear pH gradient between pH 3 and 10. The behaviour of these types of gradients in isoelectric focusing is described. Immobilized pH gradients show a number of advantages compared with carrier ampholyte generated pH gradients. The most important are: (I) the cathodic drift is completely abolished; (2) they give higher resolution and higher loading capacity: (3) they have uniform conductivity and buffering capacity; (4) they represent a milieu of known and controlled ionic strength. Key words: isoelectric focusing; immobilized pH gradients; two-dimensional techniques.

Introduction

Since the basic theoretical work of Svensson-Riibe [1.2] and the description of a simple method for the synthesis of carrier ampholytes [3], the technique of isoelectric focusing (IEF) has developed into one of the most commonly used analytical and preparative methods in work with amphoteric molecules such as proteins and peptides. The resolution obtained by analytical IEF is amongst the highest available

* Excerpts of this work were presented at the symposium 'Electrophoresis "82" (Athens, 20-24 April 1982) and at the XXX Symposium 'Protides of the Biological Fluids' (Brussels. 3-6 May 1982). 0165-022X/82/0000-0000/$02.75 ~ 1982 Elsevier Biomedical Press

318 from present biochemical separation techniques. Almost twenty years of experience with isoelectric focusing have shown that, notwithstanding the extreme success of the method, the carrier ampholyte based technique has certain inherent limitations and problems. The problem most frequently discussed in the literature is the so-called 'cathodic drift' [4] or 'plateau phenomenon' [5-7]. With this is understood the continuous slow change of what was expected to be a stable pH gradient with time. This causes problems mainly when narrow pH gradients are used, especially at high pH values, where the drift is most pronounced. In experiments using a matrix for convectional stabilization, a large part of these phenomena could be ascribed to electroendoosmosis caused by minor amounts of negative charges bound to the matrix [8], but there is also good reason to believe that it is partly due to more fundamental properties of the carrier ampholyte buffers. The equilibrium pH gradient postulated by Svensson-Rilbe [1] demands that the mass transport caused by electrical current should be balanced by diffusional mass transport. A consequence of this type of equilibrium is that a given voltage is expected to give a certain final concentration distribution of carrier ampholytes which should be independent of their initial concentration. In reality, with the conditions and time scales used in most preparative and analytical work, the resulting pH gradient is drastically influenced by variations in the initial concentration of carrier ampholytes [9]. This indicates that the equilibrium between diffusional and electrical mass transports demands appreciably longer times than the ones generally accepted in the past. Regardless of the origin of the 'cathodic drift', it is a fact that IEF as it is commonly used today is a non-equilibrium method. Although often a problem of minor importance, this sometimes limits the usefulness of the technique. With prolonged focusing times the drift might result either in depletion of carrier ampholytes in some part of the pH gradient, leading to a conductivity gap, or in an accumulation of the proteins to be separated near an electrode. In both cases it is impossible to obtain good resolution. Another effect resulting from the fact that IEF is a non-equilibrium method, is that the pH gradient reached in an experiment depends on the salt content of the sample. To avoid displacements of the pH gradient in the anodic or cathodic directions, either the salt content or the sample volume must be minimized. In flat-bed IEF runs with a number of parallel sample tracks separated by sample-free zones, the effect of high salt content, as a consequence of diffusional mass transport perpendicular to the focusing direction, can be seen as distorted zones [10]. Conventional IEF can also give curved zones for a number of other reasons, such as skewed end-electrolyte distribution, uneven cooling and poor contact between the platinum wires and the electrode strips in fiat-bed IEF. In no case are these initial disturbances ever eliminated as a result of the focusing process and, if large amounts of persulphate have been used for polymerization, disturbances even seem to be amplified [8]. In work with peptides the carrier ampholytes also introduce difficulties [11]. In preparative work it is very difficult to completely purify peptides from carrier ampholytes after the electrofocusing run [12,13]. In analytical runs the similarity of peptides and carrier ampholytes also makes the use of a number of the staining methods normally used for peptides impossible [14].

319 Another inherent weakness of the carrier ampholyte concept is that the pH gradient is generated with the aid of a large number of amphoteric compounds. With techniques available today, it can neither be guaranteed that these compounds are present in equal amounts, nor that essential properties, such as buffering capacities, are identical. There is overwhelming evidence that a large number of the individual compounds in the commercial carrier ampholytes are focused in sharp zones [15]. As a consequence of this, not only the buffering capacity but also the ionic strength, which directly influences pK and pI values, might vary in a dramatic manner in the pH gradient. The schlieren pattern [16] resulting in polyacrylamide gels containing focused carrier ampholytes gives a clear indication of the varying conditions in the pH gradients. As seen from what has been described above, present-day IEF techniques show certain limitations and problems, which call for a complementary technique which eliminates the above-discussed drawbacks. Different possibilities for creating stable pH gradients without carrier ampholytes have also been tried [17-20]. Until now no work, except the patent by Gasparic et al. [21], has been published on what might seem the most obvious possibility, namely to bind the buffering groups generating the pH gradient to the matrix used for convectional stabilization. There is probably a number of reasons for this. It might, for example, seem unrealistic that a gradient of this type would provide sufficient conductix~ity during the IEF process; moreover, bound charges could generate electroendoosmosis and the ion-exchange character of the matrix might lead to binding of proteins. On the contrary, we have found that the demand on electric conductivity is relatively small and that neither electroendoosmosis nor binding creates any problems, and thus any matrix suitable for generating the necessary convectional stabilization in IEF could be modified to generate an immobilized pH gradient suitable for IEF. The present paper describes the generation and use of immobilized pH gradients made with the aid of Immobilines. These are a number of buffering acryloyl monomers developed for the purpose of immobilizing pH gradients in polyacrylamide gels. These monomers are weak acids or bases with defined and known pK values which can be used to generate two buffer solutions with different pH values containing also the other chemicals necessary to generate a polyacrylamide gel. These two solutions are linearly mixed to generate a linear pH gradient in the desired pH interval. Provided that an electrical field with correct polarity is applied to gels containing insolubilized pH gradients, species isoelectric within the pH range of the gel will focus while other ionized compounds will collect at the electrodes. This work shows that the use of Immobiline-based pH gradients not only solves the problems associated with pH drift and distorted protein bands, but also allows IEF under controlled conditions with increased resolution and loading capacity compared to what can be achieved with carrier ampholyte-generated pH gradients.

320 Materials and Methods

Apparatus The IEF experiments were carried out using the LKB Ultrophor electrofocusing unit together with the LKB 2197 Constant Power Supply and for cooling the LKB 2209 Multitemp. For pH measurements, a LKB 2117-111 surface pH electrode was used, together with a Beckman model 3500 digital pH meter.

Chemicals Immobilines, acrylamide, N,N'-methylene bisacrylamide (Bis), ammonium persulphate, N,N,N',N'-tetramethyl ethylene diamine (TEMED), Repelsilane and Coomassie Brilliant Blue R-250 were all from LKB Produkter AB. Sulfosalicylic acid and trichloroacetic acid (TCA) were from Merck AG. All standard proteins used were purchased from Sigma. All other chemicals were of analytical grade.

Gel casting The gel casting has been made according to G6rg et al. [22] using the LKB 2117-901 gradient gel kit. The gels were 250 X 110 × 0.5 mm and, for sample application, they contained slots of the size 5 X 2 × 0.2 mm. Before use, the U-frame with the slots was coated with Repelsilane to avoid that the gel could stick to the glass surface when opening the cassette. Acrylamide and Bis have been used as separate stock solutions with the concentrations 29.1% (w/v) and 0.9% (w/v), respectively. The chambers of the gradient mixer were filled with 7 ml of each solution containing acrylamide and Bis corresponding to a 5%T.3%C gel [23] and lmmobilines in concentration adjusted to give the desired pH gradient. The acidic solution was always chosen as heavy solution and contained 20% (v/v) glycerol. The gel solutions were not degassed as the gradient mixer was used with both chambers open. The catalysts (5 /tl T E M E D and 5 ~1 of 40% persuiphate per chamber) were added directly to the gradient mixer immediately before filling the gel in the cassette. The gels were polymerized for 1 h at 50°C [24]. After removing the gels from the cassette they were placed in one liter of distilled water for half an hour whereupon excess water was allowed to drain from the vertically standing gel for another half hour at room temperature. The drying step is essential as a gel containing too much water will 'sweat' during focusing, causing small droplets to form on the gel surface. These droplets result in blue spots when the gel is stained as described below.

Running conditions In most experiments the electrodes were applied directly on the gel surface, or alternatively 10-2M H3PO 4 and NaOH could be used as electrode solutions. The samples were applied without pre-running, with the power set at 10 W, the voltage at 2500 V and the current at 25 mA. Focusing was continued overnight at 10°C for any given pH range selected. With pH values higher than 7, two strips of adsorbent cloth 200 X 20 x 5 mm were soaked in I M NaOH and placed inside the safety lid of the 2217 Ultrophor chamber in order to minimize the influence of CO 2. The gels were fixed, stained and destained as with conventional carrier ampholyte PAG-plates using Coomassie Brilliant Blue R-250 [25].

321

Two-dimensional electrophoresis The second-dimension run was done in a 360 ~m slab containing an acrylamide gradient from 4% to 22.5%T, with constant 2.5%CBi ~, 0.1% SDS and 375 mM Tris-HCl, pH 8.8. After fixing and staining, the IEF strip was washed in water for 10 min and then equilibrated for 30 to 60 min in 1% SDS, 2% fl-mercaptoethanol and 375 mM Tris-HCl, pH 8.8. The run lasted 3 h at 10°C, 50 mA, 30 W (max) and 600 V (max). For handling of gel strips, i.e. transfer from the first-dimension to the second-dimension run, see Refs. 22 and 26.

Zymograms Staining for trypsin inhibitor after IEF in Immobiline gradients was performed essentially as described by Uriel and Berges [27].

General physico-chemical properties of lmmobiline The Immobilines TM are acrylarrfide derivatives with the general chemical formula (where R contains either a carboxyl or tertiary amino group). CH 2 = C H - - C - - N - - R

JI

I

0

H

Provided that the correct polymerization conditions are chosen, these monomers are efficiently incorporated in the gel. As a result of their presence, the gel (in the absence of other compounds taking part in protolytic equilibria) will have a pH defined by the concentrations and dissociation constants of the Immobilines. The gel will also have a conductivity not only related to H* and O H - , but also to the amount of incorporated Immobilines and their buffering capacity. Table i lists the relevant physico-chemical data on Immobiline: at present three acids and four bases are available, with pK values spanning the pH range 3.6-9.3. Immobiline pK 9.3 has been used as counter-ion for determination of the pK values of the acids; conversely, Immobiline pK 3.6 has been used for the pK determination of the bases, pK measurements in gels were performed by including 2 . 1 0 2M buffering Immobiline and 10- 2 M lmmobiline counter-ion in the solution used to generate a gel of the size 240 × I10 × 1 ram. For catalyst removal, the gel was electrolyzed on the Ultrophor overnight and then the pH was measured. All pH measurements were made with a glass surface electrode calibrated with NBS standard buffers. The distance between the double bond and the group taking part in the protolytic equilibrium has in all cases been chosen long enough to ensure that the influence of the double bond on the dissociation constant is negligible. As a result, the pK difference between the Immobiline monomer and the buffering group fixed in the immobilized pH gradient when used for IEF is mainly due to media effects and temperature variations.

OF IMMOBILINES

TM

4.60

I m m o b i l i n e p K 4.6

4.61

3.58 4.39

25°C

pK pK pK pK

6.2 7.0 8.5 9.3

6.41 7.12 8.96 9.64

6.23 6.97 8.53 9.28

6.21 7.06 8.50 9.59

+ ' ~ ~

0.05 0.07 0.06 0.08

4.51 ÷ 0.02

4.30 ' 0.02

10oc

6.15 6.96 8.38 9.31

÷ ' = :

0.03 0.05 0.06 0.07

4.61 = 0.02

4.36-0.02

25°C

P o l y a c r y l a m i d e gel b T=5%, C=3%

6.24 '- 0.07 6.95 ~ 0.06 8.45 2 0.07 9.30~0.05

9.57 " 0.06

3.75 ÷ 0.02 4.47 '- 0.03 4.71 " 0.03

6.32--v. 0.08 7.08 ' 0.07 8.66 :~ 0.09

3 . 6 8 - 0.02 4.40 - 0.03 4.61 - ' 0 . 0 2

10oc

250( `

P o l y a c r y l a m i d e gel b T = 5~,, C = 3 % glycerol 25e~ ( w / v )

p K values m e a s u r e d with glass surface electrode w i t h o u t any corrections. b M e a n values of 10 d e t e r m i n a t i o n s . D u e to the slow r e s p o n s e of the electrode the p K values for the a m i n e s are uncertain.

Immobiline Immobiline Immobiline Immobiline

Bases with tertiary amine as buffering group

3.57 4.39

l m m o b i l i n e p K 3.6 I m m o b i l i n e p K 4.4

Acid~ with carboxyl as buffering group

10°C

H 2°

A p p a r e n t p K values a, / = 10 2

PROPERTIES

TABLE 1

solid liquid liquid

solid

solid solid solid

Physical state at room temperature

323 Theory In order of generate reproducible pH gradients suitable for IEF with the aid of lmmobilines, using linear gradient mixing, the following criteria have to be met: (a) the pH gradient should preferably be linear; (b) the buffering capacity must be sufficiently high to render the pH gradient insensitive to impurities (e.g. acrylic acid) and to limited accuracy in the preparation of starting solutions; (c) the buffering capacity (and conductivity) should also be even in order to minimize the effect of small disturbances in the mixing and casting of the gel. We have developed a theory for generation of linear, relatively narrow, pH gradients in the following three cases: (1) using a single buffering Immobiline centered at its pK; (2) using a single buffering Immobiline with midpoint of the pH interval removed from the pK; (3) linear mixing of two buffering Immobilines. Other combinations are possible (e.g. non-linear pH gradients, using three or more Immobilines for extended pH gradients) but these cases will be dealt with in future publications. (1) p H gradients obtained with one buffering Irnmobiline centered at its p K

The theory here is straightforward: the Henderson-Hasselbalch equation gives the relationship between the pH in the gradient and the dissociation constant (pK) of the Immobilines used. If only two lmmobilines are used, one of which is buffering in the desired pH range and the other can be regarded as fully ionized (non-buffering species), the pH can be directly calculated from the Immobiline concentration ratios and the pK of the buffering species. If the buffering Immobiline is an acid and CA and C a are the molar concentrations of acidic and basic (i.e. non-buffering) Immobilines, respectively, the pH is given by the following relationship:

ca

(1)

pH = pK A + log CA _ Ca

while in the case in which the buffering lmmobiline is a base, the corresponding expression is: C s - CA pH = pK a + log - CA

,~.'?

(2)

If the buffering Immobiline concentration is kept constant, the pH gradient resulting from linear gradient mixing of two solutions, titrated to the extremes of the desired pH interval, will correspond to an ordinary titration curve. The best pH gradients, with respect to Unearity and buffering capacity, will in this case be those centered at the pK of the buffering group. When using only one buffering species, gradients as wide as 1.2 pH units can be generated, centered at the pK, with only small deviations from linearitv.

324

(2) pH gradients obtained with one buffering lmmobiline with midpoint of the pH interval removed from the p K If only linear pH courses could be generated centered at the pK of the buffering group, the technique would be rather limited. Varying also the buffering lmmobilinc concentration will give pH gradients with inflection points at pH values differing from the pK. From the Henderson-Hasselbalch equation it is clear that a certain difference between a pH value and the p K defines a molar ratio between buffering and non-buffering lmmobiline. If pH 0 is the pH value at the new inflection point (or midpoint of the desired pH range), and if the concentrations of the acidic and basic lmmobilines at pH 0 are CA(0) and CB~0r respectively, the ApH, in relation to the midpoint, will be given by the expression: pH = p K - pH o + log

CB~o)+ bx Ca(o}- cr3(m + ( a - b )x

(3)

if the buffering lmmobiline is an acid, while, if the buffering Immobiline is a base, the corresponding relation derived from Eqn. 2 will hold. In this expression, x is the distance from the inflection point and a and b are ( d C A ) / ( d x ) and ( d C u ) / ( d x ) , respectively. From the criterion that ( d 2 p H / d x 2) = 0, it is found that, in order to convert pH 0 into the new inflection point of the function pH = f(x), the following relation should be satisfied: = ~- 2 - ~

(4)

where the negative sign results when the buffering lmmobiline is a base. With the aid of this relation it is possible to generate any linear narrow pH range in the interval 3-10 with the available Immobilines. Fig. 1 gives an example of these concepts: curve a represents the titration curve of a buffering species with midpoint centered on its pK (pH 0 - pK = 0); b and c are new curves with inflection points centered on pK + 0.3 pH units and pK + 0.6 pH units, respectively; d and e are the equivalent, symmetrical curves centered at p K - 0 . 3 pH units and p K - 0 . 6 pH units, respectively. The boxed area envelops those portions of the different titration curves which develop a linear pH gradient.

(3) pH gradients obtained with two buffering Immobilines As an example, we have chosen two basic buffering lmmobilines, B t and B2, having pK values designed as pKat and pKa2, respectively, whose concentrations are varied in a reciprocal, linear fashion (i.e. d C m / d x = --dCE~Jdx) in order to give a pH gradient whose midpoint is centered between the two pK values (i.e. pH 0 = (pK m + pKa2)/2 and Cat = Ca2 at pH0). The following equation describes the relation between pH and the distance (x) from midpoint based on the degree of ionization [ p H - pK] and on linear concentration gradients of the two buffering

325

3¢ Q. I Z O.

distance

(arbitrary

units)

Fig. I. Examples of different pH gradients obtained with a single buffering immobiline with midpoint of the pH interval removed from the pK. Curve a is the titration curve of the buffering species with midpoint centered on its p K (pH o - p K =0): b and c are new curves with pH 0 centered on p K +0.3 pH units and p K + 0.6 pH units, respectively; d and e are the equivalent, symmetrical curves centered at pK - 0 . 3 pH and p K - 0 . 6 pH units, respectively. Within this range (pH o + A p H of 0.6) linear pH gradients can still be obtained (boxed area).

Immobilines and one non-buffering counter ion: CBI + a x

lO (pH-pKR') + I

+

( CB2 -- ax ) IO (pA"~ pH) Io(PKR2 -pll} +

I

= C , + bx

(5)

where the following notations apply: x = distance from midpoint (taken as the arithmetical mean of the two pK values); b = ( d C B l ) / ( d x ) = - ( d C B 2 ) / ( d x ) (i.e. concentration gradients of the two buffering species); CA =- concentration of non-buffering acidic Immobiline when x - - 0 , and a-(dCA)/(dx) (i.e. concentration gradient of non-buffering counter ion). Eqn. 5, and the condition that the points with the pH values pKm, (3pKBI + pKB2 )/4 and (pKBj + p K B 2 ) / 2 fall on a straight line, give pH gradients which closely approximate linearity.

326

(132)

~I)

CB 2

115

2¢3 Co O. I

z

1-

CB1 ~

.T e.,

_

--1

j

O-

p%~

(arbitrary units) --15

2~

-1

"~ ~ ~. C B 1

I

I-

/

-10

-0

O. I

5 I I I

a

distance

,¢m

-

/ /

O0

f

-r

O.

I

5

0-

I I

--1

(B1) (B2) distance (arbitraryunits) Fig. 2. pH gradients obtained with two buffering lmmobilines. Two basic species. B I and B2, having pK values designated as pKnl and pKt~2 and concentrations C?Bi and CR2, have been chosen. Simple cases are demonstrated in which ('B, and Ctu are varied in a reciprocal, linear fashion to generate a pH gradient whose midpoint is centered between the two pK values (i.e. pH~ =(pKul +pKB2)/2 and C'nl = C'R2 at pl-t(:~ ). In panel a, ApK = 1.6, while in b. ApK =0.7. The solid, heavy line is the pH gradient. E x a m p l e s of p H g r a d i e n t s c r e a t e d by these m e a n s are g i v e n in Fig. 2a ( A p K = 1.6) a n d Fig. 2b ( A p K = 0.7). N o t i c e that in Fig. 2a a linear p H g r a d i e n t s p a n n i n g 2 p H u n i t s is p r a c t i c a l l y o b t a i n e d .

Results T h e f o l l o w i n g are e x a m p l e s of s o m e results o b t a i n e d with this n e w t e c h n i q u e . T h e y are by no m e a n s e x h a u s t i v e , r a t h e r they are m e a n t to give a g l i m p s e of the a m a z i n g p o s s i b i l i t i e s of this n e w m e t h o d .

327 Conductivity

The initial conductivity of an Immobiline pH gradient gel is determined by the amount of free, not covalently bound, ions in the gel. This is also true when the gel has been washed since a matrix containing lmmobiline will function to some extent as an ion exchanger. Thus, ions from the polymerization catalyst, and trace amounts of Immobiline, cannot be completely washed out from the gel with distilled water. When the unbound ions leave the gel, the conductivity will fall dramatically; this ion transport can be visibly followed by refractile lines moving towards the anodc a n d / o r the cathode marking the rear border of compounds transported towards the electrodes. If the gel initially contains large amounts of free ions, the ion transport is connected with a visible electroosmotic transport of water within the gel resulting in the build-up of a ridge towards one of the electrodes. With the Immobiline concentrations used in this work (ca. 10 mM), the conductivity falls to values in the region 0 . 2 - 2 . 1 0 6 f~-~.cm i for pH gradients in the middle of the pH scale. This is about 100 times lower than the conductivity in a conventional carrier anapholyte pH gradient [13]. This extremely high resistance means that H " and OH start contributing to the conductivity already around pH 5, on the acidic side, and pH 9, in the basic region: below and above these values the conductivity will increase sharply. In reality this does not seem to reduce the possibility of focusing in narrow gels covering a width of up to 1 pH unit in this region, as clearly shown in Fig. 3. Albeit most of the ovalbumin zones focus between pH 4.3 and pH 5.0, they all appear as extremely sharp bands, even in the low pH region where the high proton conductivity should result in a rather low field strength. Band evenness

As seen from Fig. 3, the present technique gives straight iso-pH lines covering thc whole width of the gel. In order to obtain this type of result it is essential that the pH gradient is approximately linear and the buffering power even. The results shown in Fig. 3 are also typical in that no distorted protein bands result when using lmmobiline gels. This will be true also when the sample contains large amounts of salt. If the salt content in the sample is high and variable, protein bands from samples with high conductivity will spread out over an appreciably wider part of an lmmobiline gel than what corresponds to the width of the application zone. Thus, when running this type of sample it is important to use individual gel strips for each sample track. Fig. 4 shows another advantage of immobilized pH gradients: lack of distortion in proximity of the application point. When samples are applied in pockets precast in the gel, pH distortions, due to uneven conductivity, are seen in the gel up to 2 cm above the pocket (they usually result in strongly arched bands) during conventional IEF. In the present case, even though the sample slots have been bored as circular holes through the gel thickness with a gel puncher, perfectly straight protein zones are seen throughout.

328

I~'04

Fig. 3. Varying amounts of ovalbumin focused in an Immobiline gradient ptl 4.2-5.2. Immobilinc concentrations used for gradient mixing: acidic solution: 5.10 - 3 M Immobiline pK 4.6 and 1.4.10 3 M Immobiline pK 9.3; basic solution: 5.10 - 3 M immobiline pK 4.6 and 4- 10 ~ M Immobiline pK 9.3. Running conditions: overnight at 250 V/cm and 10°C. Staining as in Ref. 25.

Resolution

T h e b a n d w i d t h a n d r e s o l u t i o n o b t a i n a b l e in I m m o b i l i n e p H g r a d i e n t s are c o m p a r a b l e to c o n v e n t i o n a l c a r r i e r a m p h o l y t e p H g r a d i e n t s , T h u s n a r r o w e r p H g r a d i e n t s d e c r e a s e the b a n d s h a r p n e s s , but the r e s o l u t i o n is increased. Fig. 5 s h o w s the results of e l e c t r o f o c u s i n g o v a l b u m i n in a c a r r i e r a m p h o l y t e p H g r a d i e n t w i t h an a p p r o x i m a t e s l o p e of 0.2 p H u n i t s / c m a n d in l m m o b i l i n e p H g r a d i e n t s with the

carbonic anhydrase

t

~'lactoglobuli~ i

, ",d

,

i

ib

,q

ovalbumln

k

Fig. 4. IEF in an immobilized pH gradient (pH range 4.3-6.7) of a mixture of carbonic anhydrase, /3-1actoglobulin and ovalbumin. The cathode is uppermost. The samples have been applied in holes bored through the gel thickness with a gel puncher, at the anodic side. All other conditions as in Fig. 3.

329

4-

A 0.2 p H / c m 140 V l c m

B 0.1pH/cm 250Vlcm

C 0.02pH/cm 500 V / c m

D 0.01pH/crn 1000 V / c r n

Fig. 5. Ovalbumin focused on a narrow Ampholine pH gradient (A) and on Immobiline gradients with varying pH slopes (B-D). Strips B-D contain 5.10 3 M ImmobilinepK 4.6 titrated with ImmobilinepK 9.3 to the respectivepH slopes. Ovalbuminload in the sample tracks (from left to right): 40, 20 and 20 ~ag. following slopes: 0.1, 0.02 and 0.01 pH units/cm, respectively. In the two last experiments, parts of the gels were cut off and stained after 16 h in order to localize the position of the main band. After 18 h the distance between the electrodes was shortened to 5 and 2.5 cm, respectively, to allow supply of 500 V / c m to one gel and 1000 V / c m to the other. The experiments were then continued for 3 more hours. In going from 0.1 pH u n i t s / c m to 0.02 pH units/cm, the ovalbumin bands are resolved into doublets. With the slope 0.01 pH units/cm, the distance between the bands in the doublets is approximately 2 mm, which should correspond to a p / d i f f e r e n c e of 0.002 pH units. From this we estimate that it should be possible to resolve bands differing by as little as 0.001 pH units. From the early work of Vesterberg and Svensson [28] the resolution limit for carrier ampholyte gradients has often been given as 0.02 pH units. In reality even slightly higher resolution is possible as long as the pH drift and conductivity gaps do not interfere, if narrow pH ranges and high field strengths are used. As an example, Allen et al. [29] have claimed the resolution of al-antitrypsin bands to be of the order of only 0.005 pH units in a narrow Ampholine pH gradient (albeit these findings have not been substantiated by Charlionet et al.) [30]. Another example of the resolution obtained by immobilized pH gradients is shown in Fig. 6. Hemoglobins (Hb) C and A 2 have a A p l of less than 0.01 pH units [31] and, in conventional pH 6 - 8 Ampholine gradients, they are spaced apart by less than 1 mm, rendering their densitometric evaluation a rather difficult task. By the use of non-linear pH gradients (Fig. 6B) [32] the separation is greatly improved, but H b C appears as a rather diffuse band. Excellent resolution, with maintenance of band sharpness, is obtained in immobilized pH gradients (Fig. 6C).

330

A

B

lorn

.lcm

•

C

A2

C

+. 2 5 m M H i s

control

•

•

.'~:..j:..

:'.!i~,

:

.

•

..~::;:~":~.z:~,~-

.

.. ,~ .,.:.. : ... . . . . .

:

•

"

.

:

.

•

. .

.' ..i...:.?:=.:

.

•

~:- ~; .. ~":~-~,_..~.,,~;:]2-

•

....~ • ._.

Fig. 6. I E F o f H b C a n d H b A 2 in a p H 6 - 9 g r a d i e n t as s u c h (A), o r w i t h 25 m M h i s t i d i n e (B), as c o m p a r e d w i t h a n I m m o b i l i n e p H g r a d i e n t ( p a n e l C) s p a n n i n g a n o m i n a l p H 7 . 7 - 8 . 2 r a n g e . H b C w a s f r o m a h o m o z y g o u s p a t i e n t w h i l e H b A 2 w a s p u r i f i e d c h r o m a t o g r a p h i c a l l y f r o m a d u l t b l o o d . In all t h r e e experiments the cathode was uppermost.

The possibility of 'blowing up' an overcrowded region of an IEF profile is one of the typical advantages of Immobiline pH gradients. This example is illustrated in Fig. 7. Panel A shows the IEF pattern of different legume species in a conventional Ampholine pH gradient. Since most proteins focus in the acidic zone, especially in the pH 5-6 interval, as also generally demonstrated for all protein species [33], we have expanded this range by using Immobilines covering first a narrow pH 4.7-6.0 interval (Fig. 7, panel B) and then a narrower pH 5.2-5.8 region (panel C). This last, very narrow pH range separates the most interesting part of the legume proteins with the highest charge heterogeneity and clearly shows species-specific patterns which are not readily apparent in the wider pH intervals.

331

A pH

B

C

pH

pH

,,.,9-~ M

t

2

3

4

I

2

3

4

1

2

3

4

Fig. 7. Left: IEF in 5%T, 3%C gels. containing 8 M urea and 2~ Ampholine pH 3.5-10. Running conditions: 1.5 h, 10°C, 1500 V (max). Center: IEF in Immobiline pH 4.7-6.0: prefocusing for I h, IEF overnight, 10°C, 2500 V. Right: IEF in lmmobiline pH 5.2-5.8: prcfocusing 1 h, IEF overnight, 10°C, 2500 V. pH gradients measured with a surface electrode. Gels stained with Coomassie Brilliant Blue R-250. Samples: 1, pea (Pisurn sattvum)" 2, broad bean ( Vwm faba); 3, lentil ( Lens culmarts); 4, chick pea (Citer arietinum); M, p l marker proteins from Pharmacia (wide and narrow ranges). The ground seeds were extracted with 25 mM Tris-Gly buffer, pH 8.6. The Ampholine gel was 250 p,m thin, while the two Immobiline gels were 500 p.m thick.

Loading capacity Fig. 8 s h o w s t h e e f f e c t o f l o a d i n g i n c r e a s i n g a m o u n t s o f c a r b o n i c a n h y d r a s e o n I l 0 m m l o n g , l 0 m m w i d e a n d 0.5 m m t h i c k I m m o b i l i n e p H g r a d i e n t s t r i p s . U p to

pH - 6.0

. . ~

20 - 5.0

v~

50

100

200

3100400

500

~'9

Fig. 8. Varying amounts of carbonic anhydrase focused on lmmobiline gels in the pH range 4.3-6.3. Acidic end concentrations: 10- 10 -3 M Immobiline pK 4.3 and 5. l0 -3 M Immobiline pK 6.2; basic end concentrations: 3. l0 -3 M immobiline pK 4.3 and 10. l0 -3 M lmmobiline pK 6.8.

332 500 ~g can be loaded on a gel layer of this size, which is appreciably more than what can be loaded on a carrier ampholyte gel. The low current in Immobiline pH gradients makes it possible to increase the gel thickness 5-10 times without problems with the cooling capacity. As also narrower pH ranges increase the loading capacity, it should be possible to handle more than 100 mg in one run in a gel with an area corresponding to an Ampholine PAG plate (110 × 265 mm): thus Immobiline pH gradients are clearly of interest in preparative runs. In this connection Immobiline pH gradients are also advantageous because they do not contain any other extractable components than the focused proteins. Moreover, the band position can be easily localized after focusing without any staining as ridges (and strong refractive index gradients) appear in the gel at the positions of focused proteins when large protein loads are used. It is thus very easy to cut out the parts of the gel containing the protein components of interest.

Behavior of proteins: pH gradient stability Due to the low conductivity of Immobiline pH gradients, the sample components will show a different behavior than on a carrier ampholyte pH gradient. If the sample is introduced in the pH gradient after the unbound ions have been electrolysed away, the conductivity of the sample will be high compared with the background conductivity and the proteins will stay at the application area for a long period of time, with an increased risk of precipitates. It is thus normally preferable to apply the sample without pre-running the gel, so that it can move together with the salt background present in the gel plate. When coloured proteins are used, they can be seen as sharp bands moving with the refractile line marking the rear border of an ionic compound moving towards an electrode. The protein band stops as it reaches the pH zone in the gel where it is isoelectric, becoming rather diffuse and then sharpening again as the focusing is continued. The time needed by a protein to reach its p l position depends on a number of factors: slope of its titration curve around its pl, slope of the immobilized pH gradient, voltage gradient applied, medium viscosity, etc. Once a protein has reached its equilibrium position in an Immobiline gradient, however, it will stay in this position indefinitely. Fig. 9 shows the behavior of sperm whale and horse heart myoglobins in a pH 6.5 to 8.75 Immobiline gradient. The proteins are focused in four hours and after 16 h at 250 V / c m they are still in the same position. Under these conditions most proteins in a conventional carrier ampholyte system would have drifted out in the cathodic compartment [33]. The indefinite stability of immobilized pH gradients is also clearly demonstrated in the set of experiments of Fig. 10. In panel A. ovalbumin was focused on a grafted pH gradient with the right electrode polarity. The field polarity was then reversed, whereby the protein zones were electrophoresed out of the gel. In panel C, the gel was run overnight with incorrect polarity; the electric field was then reversed and the sample applied. Ovalbumin focused in thisgel with the same band position and distribution as in panel A. Thus, not even by applying the electric field in the wrong direction is it possible to alter or influence the pH gradient.

333 8.'/5

oN

$,SO '

• ~n

.

......

~.....

Jib

.."~ i',:. • 4 h

.:

Oh

Fig. 9. Horse heart and sperm whale myoglobins, in alternate tracks, focused for varying times on an lmmobiline gel, pH 6.5-8.75. The gradient was created as follows: a mixture of 2.5.10- 3 M lmmobiline pK 6.2. 5.10 -3 M Immobiline pK 7.0 and 5.10- 3 M Immobiline pK 8.5 was titrated with immobiline pK 3.6 to the stated pH interval (pH 6.5 in the dense and pH 8.75 in the light gradient solutions).

Two-dimensional separations Two-dimensional method

techniques

have become

an extremely

powerful

fractionation

b a s e d o n c h a r g e , f o l l o w e d b y m a s s s e p a r a t i o n ( a s a r e v i e w , s e e R e f . 34 a n d

L.

A

B

C

Fig. 10. Check for pH gradient stability. Panel A: commercial ovalbumin was focus,ed in a grafted gradient from pH 4 to pH 6. Panel B: the electrode polarity was reversed, the pH gradient did not move, but the protein zones collected at the electrodes. Panel C: the gel was run overnight with reversed polarity, then the polarity was inverted and the sample applied; the results are identical with gel A. Running conditions: LKB 2217 Ultrophor chamber with LKB 2197 constant power supply; power set at 10 W, voltage at 2500 V and current at 25 mA. IEF: overnight at 10°C (LKB 2209 Multitemp), sample applied without pre-running. Staining: Coomassie Brilliant Blue R-250.

334

k' .

!

-3 't

_4

--7

~

--1o

"

MWxtO

i

i

i

I

4.7

5.0

5.5

6.0

pH

I

I

I

F

4.7

5.0

5.5

6.0

-4

Fig. 11. Ultrathin-layer horizontal high-resolution 2-D electrophoresis. Ist dimension: IEF in lmmobiline pH 4.7-6.0, in 5%T,3%C gels, 0.5 mm thick, containing 8 M urea. Running conditions: prefocusing for 1 h, IEF overnight, 10°C, 2500 V. After fixing and staining, the IEF strips were rinsed 10 min in water. equilibrated for 60 min in SDS buffer, blotted and loaded on the 2-D gel. 2nd dimension: SDS-PAGE in a 360 p.m thin pore gradient gel (4-22.5%T,2.5%C). Running conditions: 3 h, 10°C, 50 mA. 30 W max, 600 V. Left gel: broad bean; right: lentil proteins. The central two tracks are SDS patterns of a mixture of molecular weight marker proteins.

Vol. 28, No. 4, April 1982 of Clinical Chemistry). Reproducible 2-D maps, where constancy of spot position and intensity is maintained from run to run. are a prerequisite for any genetic or clinical study. In such studies the appearance of 'new' bands can be of p a r a m o u n t importance in detecting a mutation or the insurgence of a pathological condition in a patient. Conventional 2-D maps are beleaguered by the 'cathodic drift', which could alter the p l position of a spot along the first-dimensional axis. This is completely eliminated in grafted p H gradients, since the slope of the p H gradient can never vary along the separation axis. Fig. 11 gives an example of a 2-D map generated by using insolubilized p H gradients in the first dimension. U p o n repeated runs, the position of each spot in the 2-D map would never vary, as also shown by stacking several stained gel slabs on top of the other. Also, the fact that the protein spots do not show any severe tailing along their migration path from the I E F strip suggests that ion exchange with the grafted pH gradient, if any, is minimal.

Zymograms Since the p H gradient can never be washed out from the gel, we have wondered whether it could interfere with detection of a focused zone by its biological activity. It is known that a prerequisite for developing a z y m o g r a m after an I E F step is the adjustment of the p H of the gel to the p H of o p t i m u m enzyme activity [35]. As shown in Fig. 12, immobilized p H gradients seem to be fully compatible with z y m o g r a m techniques. The zones of activity of trypsin inhibitor from different legumes are completely developed and seem to behave as after conventional electrophoresis or I E F in Ampholine-generated p H gradients. Again, due to the high resolving power of very narrow p H gradients, species-specific isozyme patterns are

335

-4.7 pH -5.0 -5.2 -5.5 -5.8 -6.0 la

lb

2a

2b

3

4a

4b

5

6

Fig. 12. Trypsin inhibitor zymograms of different legume species and varieties. IEF in Immobiline pH 4.7-6.0 in 5%T,3%C gels, 0.5 mm thick, containing 8 M urea. pH gradient measured with a surface electrode. Running conditions: prefocusing l h, IEF overnight, 10°C, 2500V. Samples: Phaseolus vulgaris, sub-species communis, var. Mombacher Speck (la) and Zucker Perl Princess (Ib); subspecies nanus, var. Sotexa (2a) and var. Felix (2b): Phaseolus cocctneus (scarlet runner bean) var. Weisse Riesen (3); Psophocarpus tetragonolobus (winged bean) var. 980 (4a) and 943 (4b); 5, Glvcine max (soya); 6, Ceratonia siliqua (carob). After IEF the gel was incubated for 15 rain at 37°C in 200 ml of 100 m M phosphate buffer, pH 7.6, containing 60 mg trypsin; it was rinsed 6 times and left to incubate in a dry basin for 15 min at 37°C. The zymogram was developed by immersion in 180 ml of freshly made 50 mM phosphate, pH 7.6, containing 100 mg of Fast Blue B. To this solution, just prior to use, 20 ml of dimethylformamide containing 50 mg of N-acetyI-Dt,-phenylalanine-/~-naphthyl ester was added. Color development takes several hours. The reaction is stopped in methanol/acetic a c i d / H 20 (25 : 10 : 65. v/v).

readily apparent, as well as genetic variants within different varieties of the same legume.

Discussion A comparison of IEF in Immobiline pH gradients and in carrier ampholyte-generated pH gradients shows a number of advantages for the Immobiline gel: (a) true equilibrium method - no drift; (b) higher resolution; (c) higher loading capacity; (d) better control of form, width, ionic strength and buffering capacity; (e) possibility of generating extremely shallow pH gradients; (f) easier separation of buffering species from proteins in preparative runs; (g) insensitivity to salts and buffers in the sample. IEF with good quality carrier ampholytes is still an excellent analytical method which in most cases gives results which correspond to the demands of the user.

336

Immobiline pH gradients require longer focusing times and, although it is easy to cast a gradient gel, it is still easier to cast a carrier ampholyte containing gel or use a ready-made PAG plate. Thus, for most routine uses, a carrier ampholyte-based pH gradient is still the natural choice and Immobiline should be used mainly when its advantages are required. IEF in ultra-narrow pH gradients when very high resolution is needed is an obvious case when Immobiline pH gradients ought to be used. In order to detect the presence of minor components in a sample, it is often necessary to heavily overload major components which disturb the IEF process in carrier ampholyte pH gradients. The high loading capacity of Immobiline pH gradients makes them the natural choice also for this type of application. The situation described frequently appears in two-dimensional applications using IEF in the first dimension. The other advantages with Immobiline pH gradients, such as insensitivity to disturbances and ease of control of form and width of pH gradients. are also of major importance to 2-D techniques and thus Immobiline pH gradients should he preferred for the first dimension in 2-D runs. This is exemplified in the patterns of Fig. 7 and in the 2-D maps of Fig. 11. IEF patterns of legume proteins. especially in ultra-narrow, carrier ampholyte pH ranges, are usually diffused and distorted, due to low molecular mass, charged contaminants present in large amounts in seed extracts. On the other hand, immobilized pH gradients are insensitive to the vast amounts of phenolic compounds, sugars and salts typically present in plant proteins, so that the seed extracts can be applied as such, even in large volumes, without any need for previous treatments (see Figs. 7 and 11). The above is also true when dealing with biological fluids (e.g. urines, amniotic and cerebrospinal fluids, duodenal juice, etc.) which in general contain large amounts of salts on a highly diluted protein background. In these cases too it should be more convenient to use Immobiline pH gradient gels than to remove the salt before applying the samples to a conventional IEF gel. Due to the fact that none of the naturally occurring amino acids have side chains with groups buffering in the pH range 7.5-9.5, many proteins that are isoelectric in this range will approach their pl relatively slowly. This phenomenon can be directly visualized with the aid of electrophoretically run titration curves on prefocused Ampholine pH gradients [36]. Carrier ampholyte pH gradients in the pH range 7.5-9.5 are characterized by a fairly large pH drift, especially in the absence of urea [37]. The combination of the slower focusing and the pH drift can result in diffused bands and poor focusing also in pH gradients as wide as two pH units. In this case Immobiline gradients represent a valid alternative. Another field in which the use of lmmobiline should be of great interest is in preparative IEF. The high load capacity, the ease with which a narrow, specially designed pH gradient can be generated and the fact that the problem of separating the focused proteins from the carrier ampholytes is avoided, clearly ought to make the Immobiline pH gradient attractive for preparative work. We have calculated that. per unit of gel volume, a grafted pH gradient should have at least 10 times higher loading capacity than a conventional, equimolar carrier ampholyte gel (1% Ampholine~ 10 mM solution) [38]. Since biochemistry is not a magic process, we have been wondering what could be the reason for that. One hint has been given to

337 us by M. Bier at the Eiectrophoresis meeting in Athens (April 1982). In conventional IEF, a protein is both isoelectric and isoionic (i.e. at its p l it is truly stripped free of buffer counter-ions). The reason for this is that the buffering species are also isoelectric and, instead of interacting with the protein macroions, if any, they tend to form an inner salt. Conversely, it is conceivable that, in Immobiline gels, the protein, at its pl, is isoelectric but not isoionic, i.e. it tends to form a salt with the buffering groups grafted onto the matrix. This greatly increases the protein solubility at its p/, thus allowing much greater sample loads. We believe M. Bier is right along this line of thinking. In fact a 10 mM lmmobiline gel, around the pK of the buffering group, would have an ionic strength (1) of 5 mequiv./liter. On the other hand, an equimolar (1%) Ampholine gel, would have an I value 5 to 10 times lower (around 0.5-1 mequiv./liter) [39]. Why should it be so? Some hints have been given to us by H. Rilbe. He has always argued that the contribution to the solvent ionic strength of an amphoteric buffer might depend on the actual distance of the opposite charged groups in the molecule. Well, he might have a strong point there. In carrier ampholytes, the distance between the charged groups is barely 2 - 3 carbon atoms so that, in the truly isoelectric molecule, the opposite charges might fully neutralize each other and their contribution to I might be close to zero. Conversely, in grafted pH gradients, the distance between the charged groups is so huge that they might behave, at all practical purposes, as truly independent charges, thus fully contributing to the medium ionic strength. In fact, in a 5% gel, containing 10 mM lmmobiline, it can be calculated that, on the average, one charged group is incorporated in the polymer coil approximately every 70 acrylamide residues. Thus two charged groups are spaced apart, on the average, a good 140 to 150 carbon residues along the polymer backbone. To state that lmmobiline pH gradients revolutionize the art of 1EF is perhaps too strong, but quite clearly it is a technique with great possibilities for the future. Immobiline pH gradients are complementary to carrier ampholyte gradients and will certainly increase the usefulness of IEF as an analytical and preparative tool in biochemistry.

Simplified description of the method and its advantages Conventional IEF in carrier ampholytes suffers from the following drawbacks: cathodic drift, conductivity gaps, buffering capacity gaps and too low and uncontrolled ionic strength. A set of acryloyl monomers containing buffering groups (either carboxyls or tertiary amino groups) with pK values spanning the pH range 3.6-9.3 has been developed. During polymerization of polyacrylamidegels the buffering species (Immobilines)can be grafted into the matrix, thus generating pH gradients insolubilized within the gel network. Grafted pH gradients have the following advantages: (a) uniform conductivity along the separation tracks; (b) even buffering capacity; (c) defined and controlled ionic strength; (d) complete lack of 'pH drift'; (e) enormous resolution due to the possibilityof generating extremelyshallow pH gradients.

Acknowledgements We enjoyed fruitful and exciting 'brain-storming' sessions with Professors H. Rilbe (Sp~lnga, Sweden) and M. Bier (Tucson, Ariz.). Professor H. Rilbe has also

338

revised our mathematical

theory. P.G.R.

Consiglio Nazionale delle Ricerche (CNR)

and

E.G. are supported

by grants

from

and Ministero della Pubblica Instruzione

(MPI. Rome).

References 1 2 3 4 5 6 7 8 9 10 I1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Svensson, H. (1961) Acta Chem. Stand. 15. 325-341 Svensson, H. (1962) Acta ('hem. Stand. 16. 456-466 Vesterberg, O. (1969) Acta Chem. Stand. 23, 2653--2665 Righetti, P.G. and Drysdale. J.W. (1973) Ann. N.Y. Acad. Sci. 209, 163-186 Chrambach, A., Doerr, P., Finlayson. G.R., Miles, I,.E.M.. Sherins, R. and Rodbard. D. (1973) Ann. N.Y. Acad. Sci. 209. 44-69 Fawcett, J.S. (1975) in Isoelectric Focusing and lsotachophoresis (Righetti, P.G., ed.), pp. 25-37. North Holland/American Elsevier, Amsterdam Rilbe, H. (1977) in Electrofocusing and Isotachophoresis (Radola, B.J. and Graesslin, D.. eds.L pp. 35-50. de Gruyter, Berlin Righetti, P.G. and Macelloni, C. (1982) J. Biochem. Biophys. Methods 5, 1-15 Righetti, P.G. and Drysdale, .I.W. (1971) Bk)chim. Biophys. Acta 236, 17-28 Jonsson, M. (1980) Electrophoresis 1, 141-149 Righetti, P.G. and Chillemi, F. (1978) J. Chromatogr. 157, 243-251 Gelsema, W.J., De Ligny, C.L., Blanken, W.M., Haner, R.J., Roozen. A.M. and Bakker, J.A. (1980) J. Chromatogr. 196, 51-58 Righetti, P.G. and H.lelten. S. (1981) J. Biochem. Biophys. Methods 5. 259-272 Gianazza, E., Chillemi, F., Gelfi, ('. and Righetti, P.G. (1979) J. Biochem. Biophys. Methods I. 237-251 Righetti, P.G. (1977) J. Chromatogr. 138. 213-215 Righetti, P.G.. Pagani, M. and Gianazza, E. (1975) J. Chromatogr. 109, 341--356 Luner, S.J. and Kolin, A. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 898-903 Rilbe, H. (1978)J. Chromatogr. 159. 193-205 Martin, A.J.P. and Hampson, F.J. (1978) J. Chromatogr. 159, 101 110 Troitski, G.V., Savialov, P.V., Kirjukhin, I.E., Abramov, U.M. and Agitsky, G.J. (1975) Biochim. Biophys. Acta 400, 24-31 Gasparic, V., Bjellqvist, B. and Rosengren, A. (1975) Swedish patent 75 140 49-1: (1978) U.S. patent 4,130,470; (1981) German patent 2. 656. 162 Gorg, A., Postel, W., Westermeier, R., Gianazza, t-. and Righetti, P.G. (1980) J. Biochem Biophys. Methods 3. 273-284 Hjerten, S. (1962) Arch. Biochem. Biophys. Suppl. I. 147-151 Gelfi, C. and Righetti, P.G. (1981) Electrophoresis 2, 220-228 Winter, A., Ek, K and Andersson, U.B. (1977) LKB Application Note No. 250 G6rg, A., Postel, W., Westermeier, R.. Gianazza, E. and Righetti, P.G. (1981) in Electrophoresis '81 (Allen, R.C. and Arnaud, P., eds.), pp. 257-270. de Gruyter, Berlin Uriel, J. and Berges, J. (1968) Nature (London) 218, 578-579 Vesterberg, O. and Svensson, H. (1966) Acta Chem. Stand. 20, 820-834 Allen, R.C., Hasley, R.A. and Talamo, R.C. (1974) Am. J. Clin. Pathol. 62, 732-739 Charlionet, R., Martin. J.P., Sesboi,i& R., Madec, P.J. and Lefebvre. F. (1979) J. Chromatogr. 176, 89- 101 Basset. P., Braconnier, F. and Rosa, J. (1982) J. Chromatogr. 227, 267.-304 Beccaria, L., Chiumello, G., Gianazza, E.. Luppis, B. and Righetti, P.G. (1978) Am. J. Hematol. 4, 367-374 Davies, H. (1975) in Isoelectric Focusing (Arbuthnott. J.P. and Beeley, J.A., eds.), pp. 97-113. Butterworths. London

339 34 35 36 37 38 39

Righetti, P.G., Gianazza, E. and Ek, K. (1980) J. Chromatogr. 184, 415-456 Gianazza, E., Gelfi, C. and Righetti, P.G. (1980) J. Biochem. Biophys. Methods 3, 65-75 Ek, K. (1981) LKB Application Note No. 319 Gianazza, E., Astori, C. and Righetti, P.G. (1979)J. Chromatogr. 171, 161-169 Bianchi Bosisio, A., Snyder, R.S. and Righetti, P.G. (1981) J. Chromatogr. 209, 265-272 Righetti, P.G. (1980) J. Chromatogr. 190, 275-282