51

3.1Principles

The use of isoelectric focusing is limited to molecules which can beeither positively or negatively charged. Proteins, enzymes and pep-tides are such amphoteric molecules. The net charge of a protein isthe sum of all negative and positive charges of the amino acid sidechains, but the three-dimensional configuration of the protein alsoplays a role (Fig. 28).

At low pH values, the carboxylic side groups of amino acids areneutral:

R–COO– + H+ fi R–COOH

At high pH values, they are negatively charged:

R–COOH + OH– fi R–COO– + H2O

3

Isoelectric focusing abbreviation: IEF

S S

S S

SS

+ 3

+ 2

+ 1

0

- 1

- 2

- 3

3 4 5 6 7 8 9 1 0 11 p H

net charge

Isoelectricpoint [pI]

Fig. 28: Protein molecule and the dependence of the net charge on the pHvalue. A protein with this net charge has two positive charges at pH 6 and onenegative charge at pH 9.

Electrophoresis in Practice, Fourth Edition. Reiner WestermeierCopyright � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-31181-5

The substances to be separatedmust have an isoelectric pointat which they are not charged.

3 Isoelectric focusing

The amino, imidazole and guanidine side-chains of amino acidsare positively charged at low pH values:

R-NH2 + H+ fi R–NH3+

at high pH values, they are neutral:

R–NH3+ + OH– fi R–NH2 + H2O

For composite proteins such as glyco- or nucleoproteins, the netcharge is also influenced by the sugar or the nucleic acid moieties.The degree of phosphorylation also has an influence on the netcharge.

If the net charge of a protein is plotted versus the pH (Fig. 28), acontinuous curve which intersects the x-axis at the isoelectric point pIwill result. The protein with the lowest known pI is the acidic glyco-protein of the chimpanzee: pI = 1.8. Lysozyme from the human pla-centa has the highest known pI: pI = 11.7.

When a mixture of proteins is applied at a point in a pH gradient,the different proteins have a different net charge at this pH value (seeFig. 1). The positively charged proteins migrate towards the cathode,the negatively charged towards the anode, until they reach the pHvalue, where they are isoelectric.

In contrast to zone electrophoresis, isoelectric focusing is an endpoint method. This means, that the pattern – once the proteins havereached their pIs – is stable without time limit. Because of the focus-ing effect sharp protein zones and a high resolution are obtained.

Isoelectric focusing is employed with great success for protein isola-tion on a preparative scale. It is, however, mainly used for the identifica-tion of genetic variations and to investigate chemical, physical and bio-logical influences on proteins, enzymes and hormones. In the begin-ning, sucrose concentration gradient columns in liquid phase wereused, whereas gel media are almost exclusively employed nowadays.

The definition of the resolving power of isoelectric focusing wasderived by Svensson (1961):

�pI ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

D d pHð Þ=dx½ �E �du=d pHð Þ½ �

r

�pl: resolution capacityD: diffusion coefficient of the proteinE: field strength (V/cm)d(pH)/dx: pH gradientdu/d(pH): mobility slope at pI

52

Many of the microheterogene-ities in IEF patterns are due tothese modifications in themolecules.

The net charge curve is charac-teristic of a protein. With thetitration curve methodexplained in chapter 3.8 it caneasily be reproduced in a gel.

It is important to find theoptimum place in the gradientat which the proteins enter thegel without any trouble, do notaggregate and at which noprotein is unstable.

The fact of the time stability ofthe pattern is not always true:carrier ampholytes pH gradi-ents drift after some time, someproteins are not – or not verylong – stable at their pI.

The book by Righetti (1983) isrecommended for further infor-mation.

Svensson H. Acta Chem Scand.15 (1961) 325–341.

�pl is the minimum pI differ-ence needed to resolve twoneighboring bands.See also: titration curveanalysis.

3.2 Gels for IEF

This equation shows how resolution can be increased:

. When the diffusion coefficient is high, a gel with small poresmust be chosen so that diffusion is limited.

. A very flat pH gradient can be used.

But it also illustrates the limits of isoelectric focusing:

. Though the field strength can be raised by high voltages, itcannot be increased indefinitely.

. It is not possible to influence the mobility at the pI.

Nowadays isoelectric focusing can also be performed in capillaryelectrophoresis equipment, but in the following only gel IEF methodsare desribed.

3.2Gels for IEF

Analytical focusing is carried out in polyacrylamide or agarose gels. Itis advantageous to use very thin gels with large pore sizes cast ontosupport films (G�rg et al. 1978).

Polyacrylamide gelsReady polymerized carrier ampholyte polyacrylamide gels and rehy-dratable polyacrylamide gels with or without immobilized pH gradi-ents (explained below) are commercially available.

The use of washed, dried and rehydrated gels has been publishedsoon after introduction of polyacrylamide for IEF by Robinson (1972),the methodology has been considerably improved by Allen andBudowle (1986). The benefits are listed at page 197.

Hydrophobic proteins need the presence of 8 to 9 molar urea tostay in solution. Because of the buffering capacity of urea, there is alight increase in the pH in the acid part of the gel. High urea contentsin the gel lead to configurational changes in many proteins and dis-ruption of the quarternary structure. The solubility of very hydropho-bic proteins, such as membrane proteins for example, can beincreased by the addition of non-ionic detergents (e.g. Nonidet NP-40, Triton X-100) or zwitterionic detergents (e.g. CHAPS, Zwitter-gent).

53

G�rg A, Postel W, Wester-meier R. Anal Biochem. 89(1978) 60–70.

These ready-made gels are allpolymerized on support films.

Robinson HK. Anal Biochem.49 (1972) 353–366.Allen RC, Budowle B, Lack PM,Graves G. In Dunn M, Ed.Electrophoresis ’86. VCH,Weinheim (1986) 462–473.

Because the gels do not co-poly-merize with the support films inthe presence of non-ionic deter-gents, it is recommended torehydrate a prepolymerized,washed and dried gel in therelevant solution.

3 Isoelectric focusing

Agarose gelsAgarose gels for isoelectric focusing have only been available since1975, when it became possible to eliminate the charges of agarose byremoving or masking the agaropectin residues in the raw material.Agarose IEF exhibits stronger electroendosmosis than polyacrylamidegel electrophoresis IEF.

Separations in agarose gels, usually containing 0.8 to 1.0 % agar-ose, are more rapid. In addition macromolecules larger than 500 kDacan be separated since agarose pores are substantially larger thanthose of polyacrylamide gels. Another reason to use agarose for IEFis: Its components are not toxic and do not contain catalysts whichcould interfere with the separation.

It is difficult to prepare stable agarose gels with high urea concen-trations because urea disrupts the configuration of the helicoidalstructure of the polysaccharide chains. Rehydratable agarose gels areadvantageous in this case (Hoffman et al. 1989).

3.3Temperature

Since the pK values of the Immobilines, the carrier ampholytes andthe substances to be analyzed are temperature dependent, IEF mustbe carried out at a constant controlled temperature, usually 10 �C. Forthe analysis of the configuration of subunits of specific proteins, li-gand bindings or enzyme-substrate complexes, cryo-IEF methods attemperatures below 0 �C are used (Righetti, 1977, and Perella et al.1978). In order to increase the solubility of cryoproteins (like IgM),agarose IEF is performed at + 37 �C.

3.4Controlling the pH gradient

Measurement of the pH gradient with electrodes is a problem sincethese react very slowly at low temperatures. In addition additivesinfluence the measurement. CO2 diffusing into the gel from the airreacts with water to form carbonate ions. Those form the anhydrid ofcarbonic acid and lowers the pH of the alkaline part. To prevent errorswhich can occur during the measurement of pH gradients, it isrecommended to use marker proteins of known pIs. The pIs of thesample can then be measured with the help of a pH calibration curve.

54

Carboxylic and sulfate groupswhich can be charged alwaysremain.

Disadvantages: Silver stainingdoes not work as well foragarose gels as for polyacryl-amide gels. In the basic area,electroendosmosis is particu-larly strong.

Incorporation of linearpolyacrylamide:Hoffman WL,Jump AA, Kelly PJ, Elanogo-van N. Electrophoresis 10(1989) 741–747.

Righetti PG. J. Chromatogr.138 (1977) 213–215.

Perella M, Heyda A, Mosca A,Rossi-Bernardi L. Anal Biochem88 (1978) 212–224.

Cryoproteins are precipitatingat low temperatures.

Marker proteins for various pHranges exist. These proteins arechosen so that they can focusindependently of the point ofapplication.

Note: Standard markerproteins cannot be used in ureagels, because their conforma-tions are changed, and thustheir pIs.

3.5 The kinds of pH gradients

3.5The kinds of pH gradients

The prerequisite for highly resolved and reproducible separations is astable and continuous pH gradient with regular and constant conduc-tivity and buffer capacity.

There are two different concepts which meet these demands: pHgradients which are formed in the electric field by amphoteric buf-fers, the carrier ampholytes, or immobilized pH gradients in whichthe buffering groups are part of the gel medium.

3.5.1Free carrier ampholytes

The theoretical basis for the realization of “natural” pH gradients wasderived by Svensson (1961) while the practical realization is the workof Vesterberg (1969): the synthesis of a heterogeneous mixture of iso-mers of aliphatic oligoamino-oligocarboxylic acids. These buffers area spectrum of low molecular weight ampholytes with closely relatedisoelectric points.

The general chemical formula is the following:

– CH2 – N – (CH2)x – N – CH2 –

(CH2)x (CH2)x

NR2 COOH

These carrier ampholytes possess the following properties:

. a high buffering capacity and solubility at the pI,

. good and regular conductivity at the pI,

. absence of biological effects,

. a low molecular weight.

Most of the commercially available solutions contain 40 % (w/v)carrier ampholytes. The product “PharmalytesTM” are produced witha different chemistry, the concentration can therefore not be speci-fied. However, they are used with the same volumes like a 40 % solu-tion.

The pH gradient is produced by the electric field. For example, in afocusing gel with the usual concentration of 2 to 2.5 % (w/v) carrierampholyte (e.g. for gradients from pH 3 to 10) the gel has a uniformaverage pH value. Almost all the carrier ampholytes are charged:those with the higher pI positively, those with the lower pI negatively(Fig. 29).

55

Vesterberg, O. Acta Chem.Scand. 23 (1969) 2653–2666.

Where R = H or–(CH2)x–COOH,x = 2 or 3

Naturally occurring ampholytessuch as amino acids andpeptides do not have theirhighest buffering capacity attheir isoelectric point. They cantherefore not be employed.

By controlling the synthesis andthe use of a suitable mixturethe composition can be moni-tored so that regular and lineargradient result.

3 Isoelectric focusing

When an electric field is applied, the negatively charged carrierampholytes migrate towards the anode, the positively charged ones tothe cathode and their velocity depends on the magnitude of their netcharge.

The carrier ampholyte molecules with the lowest pI migratetowards the anode and those with the highest pI towards the cathode.The other carrier ampholytes align themselves in between accordingto their pI and will determine the pH of their environment. A stable,gradually increasing pH gradient from pH 3 to 10 results (Fig. 29).

Since carrier ampholytes have low molecular weights they have ahigh rate of diffusion in the gel. This means that they diffuse awayfrom their pI constantly and rapidly and migrate back to it electro-phoretically: because of this, even when there are only a limited num-ber of isomers a “smooth” pH gradient results. This is particularlyimportant when very flat pH gradients, for example between pH 4.0and 5.0, are used for high resolution.

Electrode solutionsTo maintain a gradient as stable as possible, strips of filter papersoaked in the electrode solutions are applied between the gel and theelectrodes, an acid solution is used at the anode and a basic one at thecathode. Should, for example, an acid carrier ampholyte reach theanode, its basic moiety would acquire a positive charge from the me-dium and it would be attracted back by the cathode.

56

before start in the electric field

pH

separation distance

pH

separation distance

pH

separation distance

increasing pI

carrier ampholytes

Fig. 29: Diagram of the formation of a carrierampholyte pH gradient in the electric field.

The anodal end of the gelbecomes more acidic and thecathodal side more basic.

The carrier ampholytes losepart of their charge so theconductivity of the geldecreases.

The proteins are considerablylarger than the carrier ampho-lytes – their diffusion coefficientis considerably smaller – theyfocus in sharper zones.

These electrode solutions areparticularly important for longlasting separations in gelscontaining urea, for basic andfor flat gradients. They are notnecessary for short gels.

3.5 The kinds of pH gradients

The native IEF in Fig. 30 could be carried out without electrode so-lutions because a washed and rehydrated gel with a wide pH gradientwas used.

Urea IEFFor a number of samples it is necessary to avoid protein-protein inter-actions and / or to increase the solubility by adding 8 molar urea tothe sample solution and to the gel. This causes denaturation of theproteins. The separation is slower than for native IEF, because of thehigher viscosity in the urea solution. Urea IEF gels require electrodesolutions, partly because of the extended separation times. Very pureurea must be used: When it is partly degraded to isocyanate, proteinsbecome carbamylated, resulting in artifactual additional bands.

Sometimes a nonionic or zwitterionic detergent is added in orderto increase the solubility further, and to avoid crystallysation of theurea. In these cases the copolymerization of the gel and the film sup-port does not work any more.

Urea gels are not available as ready-made gels, because of the lim-ited stability of the urea in solution. But prepolymerized and driedgels on film supports can be soaked in a fresh urea- carrier ampholytesolution short before use. This procedure was, for instance, appliedon the differentiation of fish varieties by Rehbein et al.

57

Fig. 30: Isoelectric focusing in a washed and rehydrated polyacrylamide gel.Press sap of potatoes of different varieties. Coomassie Brilliant Blue staining.(Anode on top). From Pharmacia, Freiburg.

Electrode solutions for agaroseIEF are listed in chapter 5, forpolyacrylamide gels – ifrequired – in chapter 6 ofpart II.

It is recommended to prepareurea solutions fresh, andremove isocyanate from theurea solution with mixed bedion exchanger short before use.

Rehbein H, K�ndiger R,Pineiro C, Perez-Martin RI.Electrophoresis 21 (2000)1458–1463.

3 Isoelectric focusing

Separator IEFEver since the introduction of IEF, modifications of the pH gradientshave been investigated. If the resolution is not satisfactory, it is oftenpossible to add separators (Brown et al. 1977):

These are amino acids or amphoteric buffer substances which flat-ten the pH gradient in the area of their pI. Their position in the gra-dient can be changed by adapting the temperature conditions andseparator concentration, so that complete separation of neighboringprotein bands can be achieved.

One example is the separation of glycosylated HbA from the neigh-bouring main hemoglobin band in the pH gradient 6 to 8 by the addi-tion of 0.33 mol/L b-alanine at 15 �C (Jeppson et al. 1978).

Plateau phenomenonProblems with carrier ampholytes can arise when long focusingtimes are necessary. For example, when narrow pH intervals are areused, or in the presence of highly viscous additives such as urea ornon-ionic detergents, the gradient begins to drift in both directionsbut specially towards the cathode.

This leads to a plateau in the middle with gaps in the conductivity.Part of the proteins migrate out of the gel (Righetti and Drysdale,1973) and are not included. Because of the limited number of differ-ent homologues, the gradients cannot be flattened and the resolutioncapacity not increased at will.

The procedure of a carrier ampholyte IEF runAs isoelectric focusing is in principle a nondenaturing method, theoptimization of the running conditions is very important to preventprecipitation and aggregation of proteins, and to achieve good repro-ducibility.

. Temperature setting

. Prefocusing

. Sample loading

. Sample entry

. Separation time is a compromise between letting all proteinsreach their pIs and keeping the gradient drift to a minimum.

. Fixing (with TCA or by immunofixation) and staining – oralternatively – application of zymogram detection.

58

Brown RK, Caspers ML,Lull JM, Vinogradov SN,Felgenhauer K, Nekic M.J Chromatogr. 131 (1977)223–232.

Jeppson JO, Franzen B, Nils-son VO. Sci Tools. 25 (1978)69–73.

Righetti PG, Drysdale JW.Ann NY Acad Sci. 209 (1973)163–187.

A gel can “burn” through at theconductivity gaps.

The IEF running conditionsshould always be described in aprotocol or a publication.

pIs are highly dependent on thetemperature.

To establish the gradient.

On the optimized location withthe optimized mode.

At low field strength to preventaggregation.

Volthour integration is oftenused.

Proteins have to be fixed duringthe carrier ampholytes arewashed out.

3.5 The kinds of pH gradients

3.5.2Immobilized pH gradients

Because of some limitations of the carrier ampholytes method, analternative technique was developed: immobilized pH gradients orIPG (Bjellqvist et al. 1982). This gradient is built with acrylamide de-rivatives with buffering groups, the Immobilines, by co-polymeriza-tion of the acrylamide monomers in a polyacrylamide gel.

The general structure is the following:

CH2 = CH – C – N – R

O H

An Immobiline is a weak acid or base defined by its pK value.At the moment the commercially available ones are:

. two acids (carboxylic groups) with pK 3.6 and pK 4.6.

. four bases (tertiary amino groups) with pK 6.2, pK 7.0, pK 8.5and pK 9.3.

To be able to buffer at a precise pH value, at least two differentImmobilines are necessary, an acid and a base. Fig. 31 shows a dia-gram of a polyacrylamide gel with polymerized Immobilines, the pHvalue is set by the ratio of the Immobilines in the mixture.

59

C O

O

-

-

O

C O

+R

NR

+N H

R R

+

N H

R R

Fig. 31: Diagram of a polyacrylamide network with co-polymerized Immobi-lines.

Bjellqvist B, Ek K, Righetti PG,Gianazza E, G�rg A, Wester-meier R, Postel W. J BiochemBiophys Methods. 6 (1982)317–339.

R contains either a carboxylic oran amino group.

The wider the pH gradientdesired, the more Immobilinehomologues are needed.

3 Isoelectric focusing

A pH gradient is obtained by the continuous change in the ratio ofImmobilines. The principle is that of an acid base titration and thepH value at each stage is defined by the Henderson-Hasselbalch equa-tion:

pH ¼ pKB þ logCB�CA

CA

when the buffering Immobiline is a base.If the buffering Immobiline is an acid, the equation becomes:

pH ¼ pKA þ logCB

CA�CB

Preparation of immobilized pH gradientsIn practice immobilized pH gradients are prepared by linear mixingof two different polymerization solutions with a gradient maker (seeFig. 21), as for pore gradients. In principle a concentration gradient ispoured. Both solutions contain acrylamide monomers and catalystsfor the polymerization of the gel matrix.

Immobiline stock solutions with concentrations of 0.2 mol/L areused. The solution which is made denser with glycerol is at the acidend of the desired pH gradient, the other solution is at the basic end.During polymerization, the buffering carboxylic and amino groupscovalently bind to the gel matrix.

Applications of immobilized pH gradientsImmobilized pH gradients can be exactly calculated in advance andadapted to the separation problem. Very high resolution can beachieved by the preparation of very flat gradients with up to 0.01 pHunits per cm.

Since the gradient is fixed in the gel it stays unchanged during thelong separation times which are necessary for flat gradients, but alsowhen viscous additives such as urea and non-ionic detergents areused. In addition there are no wavy iso-pH lines: the gradient is notinfluenced by proteins and salts in the solution.

Recipes for the preparation of narrow and wide immobilized pHgradients are given in this book in the section on methods for immo-bilized pH gradients (part II, method 10). The quantities necessaryfor the 0.2 molar Immobiline stock solutions for the acid and basicstarter solutions are given in mL for the standard gel volume.

60

Here the pH gradient is abso-lutely continuous.

CA and CB are the molarconcentrations of the acid, andbasic Immobiline, respectively.

0.5 mm thick Immobiline gels,polymerized on a support filmhave proved most convenient.

The catalysts must be washedout of the gel because theyinterfere with IEF. This is morerapid if the gel is thin.

It has proved very practical todry the gels after washing themand to let them soak in theadditive solution afterwards.

These features are particularlyuseful for the first dimension inhigh resolution two-dimen-sional electrophoresis. The gelsare cut in narrow strips for indi-vidual sample runs (see chapter6 and method 10).

The broadest pH gradientwhich can, at present, beprepared with commerciallyavailable Immobilines encom-passes 6 pH units: from 4.0 to10.0; the broadest commer-cially available gradient spansover 8 pH units: 3–11.

3.5 The kinds of pH gradients

A very comprehensive source of informations on immobilized pHgradients is the book by Righetti (1990).

Further developmentsAltland (1990) and Giaffreda et al. (1993) have published software forpersonal computers which permit the calculation the desired pH gra-dients with optimization of the distribution of buffer concentrationand ionic strength.

In the meantime it has been possible to expand the pH range men-tioned before in both directions by using additional types of Immobi-lines and also to prepare very acidic (Chiari M et al. 1989a) and basicnarrow pH gradients (Chiari M et al. 1989b). These are an additionalacid with pK 0.8 and a base with pK 10.4. At these pH extremities thebuffering capacity of the water ions H+ and OH– must be taken intoconsideration. Furthermore dramatic differences occur in the voltagegradient, which must be compensated by the gradual addition ofadditives to the gradient.

For adequate and reproducible analysis of very basic proteins likelysozyme, histones, and ribosomal proteins in an immobilized pHgradient 9–12 several methodical modifications are necessary (G�rget al. 1997).

61

Fig. 32: IEF in immobilized pH gradient pH 4.0 to 5.0. Isoforms ofa1-antitrypsin (protease inhibitors) in human serum. By kind permission ofProf. Dr. Pollack and Ms. Pack, Institut f�r Rechtsmedizin der Universit�tFreiburg im Breisgau. (Anode at the top).

Righetti PG. Immobilized pHgradients: theory and metho-dology. Elsevier, Amsterdam(1990).

Altland K. Electrophoresis 11(1990) 140–147.Giaffreda E, Tonani C, Righet-ti PG. J Chromatogr. 630(1993) 313–327.

Acidic Immobiline:Chiari M, Casale E, Santa-niello E, Righetti PG. TheorApplied Electr. 1 (1989a)99–102.

Basic Immobiline:Chiari M, Casale E, Santa-niello E, Righetti PG. TheorApplied Electr. 1 (1989b)103–107.

G�rg A, Obermaier C,Boguth G, Csordas A, Diaz J-J,Madjar J-J. Electrophoresis 18(1997) 328–337.

3 Isoelectric focusing

The use of immobilized pH gradients is at present restricted topolyacrylamide gels only.

Denaturing gradient gel IEF: Immobilized pH gradient gels with aperpendicular urea gradient from 0 to 8 mol/L have been employedby Altland and Hackler (1984) and Jenne et al. (1996) for the separa-tion of human plasma proteins to detect various mutations, whichcause diseases. The technique is similar to DGGE described onpage 30, but is easier to perform, and – as it is done on the proteinlevel – provides more informations on the danger of a mutation.

Fig. 32 shows an isoelectric focusing result of a1-antitrypsin iso-forms in IPG pH 4.0 to 5.0.

3.6Protein detection in IEF gels

Because the proteins are present in native form, and large pore sizegels are used for isoelectric focusing, the proteins need to be fixedmuch more intensively before staining than after zone elctrophoresisseparations. Usually 20 % (w/v) TCA is used. Ammoniacal silverstaining shows a much better sensitivity for protein detection in IEFgels than all other modifications of silver staining. In native gelszymogram techniques for the functional detection of enzymes can beemployed. The zymogram techniques are also applied on titrationcurve gels (see 3.8) and work best, when no acrylamide monomersare present, like in agarose and washed and rehydrated gels.

3.7Preparative isoelectric focusing

Preparative carrier ampholyte IEF is mainly carried out in horizontaltroughs in granular gels (Radola, 1973). A highly purified dextran gelis mixed with the carrier ampholyte and poured in the trough. Herefocusing is done over a long separation distance: about 25 cm. Afterprefocusing to establish the pH gradient a section of the gel isremoved from a specific part of the gradient, mixed with the sampleand poured back into place.

Carrier ampholyte IEF

After IEF the protein or enzyme zones can be detected by staining apaper replica. To recover them, the gel is fractionated with a latticeand the fractions eluted out of the gel with a buffer. Proteins quanti-ties of the order of 100 mg can thus be isolated.

62

This means that the pore size islimited towards the top.

Jenne DE, Denzel K, Bl�tzin-ger P, Winter P, Obermaier B,Linke RP, Altland K. Proc NatlAcad Sci USA. 93 (1996)6302–6307.

Radola BJ. Biochim BiophysActa. 295 (1973) 412–428.The procedure is thoroughlydescribed in: Westermeier R: InCutler P, Ed. Protein purifica-tion protocols. Second edition.Methods in molecular biologyVolume 244. Humana Press,Totowa, NJ (2004) 225–232.

For the elution, small columnswith nylon sieves are used.

3.7 Preparative isoelectric focusing

The method has lately experienced a renaissance as a very usefultool for prefractionation of highly heterogeneous protein mixturesunder denaturing conditions for high resolution 2-D electrophoresisin narrow pH intervals (G�rg et al. 2002).

Immobilized pH gradients

Immobilized pH gradients are also very useful for preparative separa-tions:

. They offer a high loading capacity.

. The buffering groups are fixed in the gel.

. The conductivity is low, so even gels which are 5 mm thickhardly heat up.

Polyacrylamide gels with IPG bind proteins more strongly thanother media, so electrophoretic elution methods must be used (Righ-etti and Gelfi, 1984).

This technique is especially useful for low molecular peptidessince the buffering groups of the gradient stay in the gel (Gianazza etal. 1983). Peptides are the same size and – after IEF – possess thesame charge as the carrier ampholytes so they cannot be separated.

Isoelectric membranes: An important approach is the application ofthe principles and chemistry of immobilized pH gradients on highresolution separation of proteins in a gel-free liquid. Righetti et al.(1989) have designed a multicompartment apparatus, whose seg-ments are divided by isoelectric Immobiline membranes. The electro-des are located in the two outer segments. The separation happensbetween the isoelectric membranes in gel-free liquid, which is con-stantly recirculated.

The highlight of the system are the membranes with defined pHvalues (Wenger et al. 1987): glass microfiber filters are soaked inacrylamide polymerization solutions, which are titrated exactly to thedesired pH values with Immobilines. Thus “crystal grade” proteinsare obtained without further contamination. Speicher and Zhou(2000) have successfully used such an instrument for prefractionationof complex protein mixtures prior to 2-D electrophoresis.

The Fig. 33 shows the principle of the purification of a protein in athree-chamber setup, where the chambers are divided by two mem-branes with pH values closely below and above the pI of the proteinto be purified.

63

G�rg A, Boguth G, K�pf A,Reil G, Parlar H, Weiss W.Proteomics 2 (2002) 1652–1657.

Righetti PG, Gelfi C. J BiochemBiophys Methods. 9 (1984)103–119.

Gianazza E, Chillemi F,Duranti M, Righetti PG,J Biochem Biophys Methods. 8(1983) 339–351.

Righetti PG, Wenisch E,Faupel M. J Chromatogr. 475(1989) 293–309.

Wenger P, de Zuanni M,Javet P, Righetti PG. J BiochemBiophys Methods 14 (1987)29–43.Speicher DW, Zuo X. AnalBiochem. 284 (2000) 266–278.

3 Isoelectric focusing

3.8Titration curve analysis

Carrier ampholyte gels can also be used to determine the chargeintensity curve of proteins. This method is very useful for several rea-sons: it yields extensive information about the characteristics of a pro-tein or enzyme, for example the increase in mobility around the pI,conformational changes or ligand bindings properties depending onthe pH. The pH optimum for separation of proteins with ion-exchange chromatography and for preparative electrophoresis canalso be established (Rosengren et al. 1977).

A pre-run is performed in the square gel without any sample untilthe pH gradient is established. The gel, placed on the cooling plate, isrotated by 90� and the sample is applied in a long trough previouslypolymerized into the gel (see Fig. 34 A).

When an electric field is applied perpendicular to the pH gradient,the carrier ampholytes will stay in place since their net charges arezero at their pIs.

64

isoelectric membranes

pH

Sample

Anode Cathode

5.4 5.6

Protein pI = 5.5A

A

A

A

B

B

B

B

B

C

C

D

D

D

D

D

D

D

A

A

A

BB

B

C

C

C

Fig. 33: Purification of a protein (A) betweentwo isoelectric membranes. All contaminatingcharged substances and proteins with pIslower than pH 5.4 (B) or higher than pH 5.6(C, D) migrate out of the central chamber,which is enclosed by the two membranes.

Rosengren A, Bjellqvist B,Gasparic V. In: Radola BJ,Graesslin D. Ed. Electrofocusingand isotachophoresis. W. deGruyter, Berlin (1977) 165–171.

A gel with large pore sizes (4 to5 % T) is used, in order toavoid influence s of the mole-cule sizes on the mobilities.

In practice a series of nativeelectrophoresis runs undervarious pH conditions arecarried out.

3.8 Titration curve analysis

The sample proteins will migrate with different mobilities accord-ing to the pH value at each point and will form curves similar to theclassical acid-base titration curves (Fig. 34 B). The pI of a protein isthe point at which the curve intersects the sample trough.

There is a representation standard for titration curves for purposesof comparison: the gel is oriented so that the pH values increase fromleft to right and the cathode is on top (Fig. 35).

65

A B

3 4 5 6 7 8 9 10 pH 3 4 5 6 7 8 9 10 pH

Fig. 34: Titration curves. A) Application of the sample in the sample trench afterthe pH gradient has been established. B) Titration curves.

Fig. 35: Titration curves of a pI marker protein mixture pH 3 to 10. Cathode atthe top.

As can be seen, no buffer reser-voirs are necessary for nativeelectrophoresis in amphotericbuffers. This forms the basis ofan electrophoretic method,which is described in method 4.