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Part I:

Fundamentals

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

Electrophoretic separation techniques are at least as widely distribut-ed as chromatographic methods. With electrophoresis a high separa-tion efficiency can be achieved using a relatively limited amount ofequipment. It is mainly applied for analytical rather than for prepara-tive purposes. However, with the advent of new technology likeamplification of DNA fragments with Polymerase Chain Reaction(PCR�), and highly sensitive and powerful mass spectrometry analy-sis of proteins and peptides, so called “analytical amounts” of electro-phoretically separated fractions can now be further analysed.

The main fields of application are biological and biochemicalresearch, protein chemistry, pharmacology, forensic medicine, clini-cal investigations, veterinary science, food control as well as molecu-lar biology. It will become increasingly important to be able to chooseand carry out the appropriate electrophoresis technique for specificseparation problems.

The monograph by Andrews (Andrews 1986) is one of the mostcomplete and practice-oriented books about electrophoretic methods.In the present book, electrophoretic methods and their applicationswill be presented in a much more condensed form.

Principle: Under the influence of an electrical field charged mole-cules and particles migrate in the direction of the electrode bearingthe opposite charge. During this process, the substances are usuallyin aqueous solution. Because of their varying charges and masses,different molecules and particles of a mixture will migrate at differentvelocities and will thus be separated into single fractions.

The electrophoretic mobility, which is a measure of the migrationvelocity, is a significant and characteristic parameter of a chargedmolecule or particle. It is dependent on the pK values of the chargedgroups and the size of the molecule or particle. It is influenced by thetype, concentration and pH of the buffer, by the temperature and thefield strength as well as by the nature of the support material. Electro-phoretic separations are carried out in free solutions as in capillary

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Introduction

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

Andrews AT. Electrophoresis,theory techniques andbiochemical and clinical appli-cations. Clarendon Press,Oxford (1986).

Chrambach A. The practice ofquantitative gel electrophoresis.VCH Weinheim (1985).

Mosher RA, Saville DA,Thormann W. The dynamicsof electrophoresis.VCH Weinheim (1992).

Introduction

and free flow systems, or in stabilizing media such as thin-layerplates, films or gels. Detailed theoretical explanations can be found inthe books by Chrambach (1985) and Mosher et al. (1992).

Sometimes the relative electrophoretic mobility of substances isspecified. It is calculated relative to the migration distance of a stan-dard substance, mostly a dye like bromophenol blue, which has beenapplied as an internal standard.

Three basically different electrophoretic separation methods areemployed in practice nowadays:

a) Electrophoresis, sometimes called zone electrophoresis (ZE).b) Isotachophoresis or ITPc) Isoelectric focusing or IEF

The three separation principles are illustrated in Fig. 1.

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= A

= B

pH

=co

nst

mR

A

mR

B

L

T

L

T

pH

=p

H

LT

zone electrophoresis isotachophoresis isoelectric focusing

pI

A

pI

B

pH

gradient

34

56

78

910

Fig. 1: The three electrophoretic separation principles. Explanations in the text.A and B are the components of the sample.

The relative mobility is abbre-viated as mr or Rm.

There is a fourth method:“Moving Boundary Electro-phoresis”, which is described onpage 9. However this techniquehas no practical importanceanymore.“Electrophoresis” is a generalterm for all these methods.Blotting is not seen as a separa-tion, but as a detectionmethod.

Introduction

a) In zone electrophoresis a homogeneous buffer system is usedover the whole separation time and range so as to ensure aconstant pH value. The migration distances during a definedtime limit are a measure of the electrophoretic mobilities ofthe various substances. It can be applied to nonamphoteric aswell as amphoteric molecules. During the separation diffusioncan lead to blurred zones, which reduces the sensitivity ofdetection and the resolution.

b) In isotachophoresis (ITP), the separation is carried out in a dis-continuous buffer system. The ionized sample migrates be-tween a leading electrolyte with a high mobility and a termi-nating – sometimes called trailing – ion with a low mobility,all of them migrating with the same speed. The different com-ponents are separated according to their electrophoretic mobil-ities and form stacks: the substance with the highest mobilitydirectly follows the leading ion, the one with the lowest mobil-ity migrates directly in front of the terminating electrolyte. InITP there is a concentration regulating effect which worksagainst diffusion.

c) Isoelectric focusing (IEF) takes place in a pH gradient and canonly be used for amphoteric substances such as peptides andproteins. The molecules move towards the anode or the cath-ode until they reach a position in the pH gradient where theirnet charges are zero. This pH value is the “isoelectric point” (pI)of the substance. Since it is no longer charged, the electricfield does not have any influence on it. Should the substancediffuse away, it will gain a net charge again, and the appliedelectric field will cause it to migrate back to its pI. This concen-trating effect leads to the name focusing. Thus also with IEFthere is no problem with diffusion.

Areas of applications: Mainly proteins, peptides, sugars, and nucleicacids are separated. Electrophoretic methods are used for the qualita-tive characterization of a substance or mixture of substances, for con-trol of purity, quantitative determinations, and preparative purposes.The most prominent fields are the Genome and the Proteome analy-sis. The word “Proteome” was introduced by Mark Wilkins during acongress in Sienna 1994, in written form in the publication byWasinger et al. one year later.

The scope of the applications ranges from whole cells and particlesto nucleic acids, proteins, peptides, amino acids, organic acids andbases, drugs, pesticides and inorganic anions and cations – in short –everything that can carry a charge.

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This is also valid for disc electro-phoresis, because a discontin-uous system exists only at thebeginning of the separation andchanges into a homogeneousone.

In comparison to other electro-phoretic and chromatographicseparation methods, ITP isconsidered exotic because thereare no spaces between thezones: the bands are not“peaks” (Gaussian curves) but“spikes” (concentration depen-dent bands). ITP is mostlyapplied for stacking of thesamples during the first phaseof disc electrophoresis.

In IEF it is important to findthe correct place in the pHgradient to apply the sample,since some substances areunstable at certain pH values(see below).

Wasinger VC, Cordwell SJ,Cerpa-Poljak A, Yan JX,Gooley AA, Wilkins MR,Duncan MW, Harris R,Williams KL, Humphery-Smith I. Electrophoresis 16(1995) 1090–1094.

Introduction

The sample: An important criterion for the choice of the appropriateelectrophoretic method is the nature of the sample to be analyzed.There must be no solid particles or fatty components suspended inthe solution. Those interfere with the separation by blocking thepores of the matrix. Sample solutions are mostly centrifuged, some-times also desalted, before electrophoresis.

Substances which are exclusively negatively or positively chargedare easy to run: Examples of such anions or cations are: nucleic acids,dyes, phenols and organic acids or bases. Amphoteric molecules suchas amino acids, peptides, proteins and enzymes have positive or nega-tive net charges depending on the pH of the buffer, because they pos-sess acidic as well as basic groups.

Proteins and enzymes are often sensitive to certain pH values orbuffer substances; conformational changes, denaturation, complexformation, and intermolecular interactions are possible. The concen-tration of the substances in the solution also plays a role. In particu-lar, when the sample enters the gel, overloading effects can occurwhen the protein concentration reaches a critical value during thetransition from the solution into the more restrictive gel matrix.

For sodium dodecyl sulphate electrophoresis, the sample mustfirst be denatured; which means it must be converted into molecule-detergent micelles. The method of selective sample extraction, partic-ularly the extraction of not easily soluble substances often determinesthe nature of the buffer to be used.The nature of the stabilizing medi-um, e.g. a gel, is dependent on the size of the molecule to be ana-lyzed.

The buffer: The electrophoretic separation of samples is done in abuffer with a precise pH value and a constant ionic strength. Theionic strength should be as low as possible so that both the contribu-tion of the sample ions to the total current and their speed will behigh enough.

During electrophoresis, the buffer ions are carried through the geljust like the sample ions: negatively charged ions towards the anode,positively charged ones towards the cathode. This should be achievedwith as little energy as possible so that not much Joule heat is devel-oped.

With the help of the Second Law of Electrolysis by Michael Faradayit is possible to calculate the amount of ions migrating in an electro-phoresis experiment: The amounts of electricity are equal to theamount of substances, which are eliminated from different electro-lytes. Taking the Avogadro constant and the elemental charge, thismeans: 1 mAh equals 36.4 lmol.

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Sample application on gelswhich are immersed in buffer(e.g. vertical and submarinegels) is done with syringes intosample wells polymerized intothe gel or into glass tubes, thesample density is raised withglycerol or sucrose.

In “Proteome analysis”, wherecomplex mixtures of severalthousand proteins have to beseparated in one gel; thesample preparation proceduregreatly influences the result.

For open surfaces as in hori-zontal systems (e.g. celluloseacetate, agarose gels and auto-mated electrophoresis) eithersample applicators are used, orthe sample is pipetted intosample wells with a micropip-ette. Capillary systems usuallyhave an automated sampleapplicator.

Yet a minimum buffering capa-city is required so that the pHvalue of the samples analyzeddoes not have any influence onthe system.

Elemental charge:1.602 � 10–19 As;Avogadro constant:1.602 � 1023 elemental unitsper mol.

Introduction

To guarantee constant pH and buffer conditions the supplies ofelectrode buffers must be large enough. The use of buffer gel stripsor wicks instead of tanks is very practical, though only feasible in hor-izontal flatbed systems.

In vertical or capillary systems, the pH is very often set to a very high(or low) value, so that as many as possible sample molecules are nega-tively (or positively) charged, and thus migrate in the same direction.

When a gel matrix does not contain any ions from polymerization,amphoteric buffers can be applied, which do not migrate during elec-trophoresis. Such a buffer substance, however, must possess a highbuffering capacity at its isoelectric point. For some applications, nobuffer reservoirs are necessary with this method.

Electroendosmosis: The static support, the stabilizing medium (e.g.the gel) and/or the surface of the separation equipment such as glassplates, tubes or capillaries can carry charged groups: e.g. carboxylicgroups in starch and agarose, sulfonic groups in agarose, siliciumoxide on glass surfaces. These groups become ionized in basic andneutral buffers: in the electric field they will be attracted by theanode. As they are fixed in the matrix, they cannot migrate. Thisresults in a compensation by the counterflow of H3O+ ions towardsthe cathode: electroendosmosis.

In gels, this effect is observed as a water flow towards the cathode,which carries the solubilized substances along. The electrophoreticand electroosmotic migrations are then additive (see Fig. 2). Theresults are: blurred zones, and drying of the gel in the anodal area offlatbed gels.

When fixed groups are positively charged, the electro-osmotic flowis directed towards the anode.

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Fig. 2: Electroendosmosis: Negatively charged groups fixed to the gel matrix orto a surface cause the flow of water ions. This results in a water transport intothe opposite direction of the electrophoretic migration of the sample ions,leading to blurred band pattern.

For anionic electrophoresis verybasic, and for cationic electro-phoresis very acidic buffers areused.

In these systems the sample isloaded at one end of theseparation medium.

A polyacrylamide gel, which iscovalently bound to a plasticfilm can be washed after poly-merization. See method 4 inthis book.

Electroendosmosis is normallyseen as a negative effect,yet a few methods takeadvantage of this effect toachieve separation or detectionresults (see page 12: MEKC andpage 19: counterimmunoelectrophoresis).

In capillary electrophoresismostly the term ”electroos-motic flow” is applied, the term”electroendosmosis” is onlyused in gel electrophoresis.