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5.1Introduction

5.1.1Purity control

To control purity, electrophoretic methods are usually used in combi-nation with chromatography. The physico-chemical properties of thesubstances to be investigated are generally known.

SDS electrophoresis is the most frequently used technique for proteinanalysis. During SDS electrophoresis, configurational differences andirrelevant charge heterogeneities of polypeptides or enzymes are elimi-nated so that only real impurities appear as extra bands of different mo-lecular weights. For analysis of low molecular weight peptides specialdesigned gels or buffer systems are usually applied. In some cases theload of the major fraction has to be very high, because low amounts ofcontaminating proteins must be detected and – sometimes – quantified.

When the kind of glycosylation or the charge properties of a pro-tein are significant, isoelectric focusing is used. In some cases bandheterogeneity caused by the various conformations of a moleculemust be taken into account when interpreting results.

Agarose submarine gels are often used for nucleic acids. Ethidiumbromide staining is used for detection, either with hybridization inthe gel or after blotting on an immobilizing membrane.

5.1.2Quantification prerequisites

Quantification is either carried out with direct UV measurement ofthe zones in capillary systems or – when support materials such asgels or films are used – indirectly by autoradiography or staining ofthe zones followed by densitometric measurement.

5

Interpretation of electropherograms

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

Gel electrophoresis has theadvantage that a large numberof samples can be analyzed atone time.

Depending on the type anddegree of impurity, stainingmethods with high or low sensi-tivity are employed; blottingtechniques are used for manyexperiments.

In cases of very high proteinloads, a complete discontinuousbuffer system has to be applied(see page 34).

5 Interpretation of electropherograms

In capillary electrophoresis, the scans resemble chromatogramsand the peaks can be integrated as in standard chromatography orHPLC.

In immunoelectrophoresis, the distance between the precipitationline and the origin is a measure of the concentration of the substancein the sample, regardless of whether the process involves electropho-retic or electroosmotic migration or diffusion.

It is, however, important to know the quantity of antibody intro-duced as well as the titre of the antigen-antibody mixture. The clearestand most reproducible results are obtained with the rocket method ofLaurell (1966). The areas enclosed by the precipitation lines are propor-tional to the amount of antibody in the sample. In many cases, it isaccurate enough to simply measure the height of the precipitin arc.

The success of quantitative determinations using electrophoreticseparations on gels or other supports depends on several factors:

. During sample preparation, substance losses through adsorp-tion on membranes or column material during desalting orconcentration should be avoided as well as the formation ofirreversible deposits during extraction or precipitation. Com-plexing or chelating agents must also be removed from thesample or else complex formation must be inhibited.

. The application method must be chosen in such a way that allsubstances completely penetrate the separation medium. Thisis especially critical during isoelectric focusing of heterogeneousprotein mixtures since different proteins are unstable at differ-ent pH values or have a tendency to aggregate. In such cases itcan be assumed that all proteins will not penetrate the mediumat the same point. During sample application, the volumetricprecision of the syringe or micropipette is also important.

. The quality of the separation is crucial for densitometric mea-surements. Wavy distorted zones, which result for examplefrom the salt concentration being too high, lead to question-able densitograms. In addition, a zone can only be properlyquantified when it is well separated from the neighboringbands.

. The prerequisite for a reliable quantification is an effectivestaining of the bands while avoiding destaining during back-ground washing.

Nevertheless, it should always be presumed that differences instaining effectiveness exist. For this reason a protein mixture consist-ing of known pure substances (e.g. marker proteins) at various con-centrations (serial dilutions) should always be applied and run in par-allel as is done in qualitative comparison of bands for molecularweight or isoelectric point determination.

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The quality of the resultsdepends on the quality of theantibodies: impurities withcross-reacting antibodies mustbe eliminated.

For practical reasons, methodsfor sample preparation aredescribed in connection witheach separation method.

Qualitative differences duringseparations can be compen-sated with the help of markerproteins.

Hot staining and colloidalstaining methods are recom-mended.

5.2 Image analysis

Since each protein possesses a specific affinity to the dye usedwhich is different from other proteins, it should always be remem-bered that only relative quantitative values can be determined.

Gradient gels almost always display an increasingly or decreasinglyshaded background after staining. This should be accounted for dur-ing photometric measurement of the electrophoresis bands and theensuing integration of the surfaces.

5.2Image analysis

In many cases it is sufficient to compare the bands and spot patternsvisually, to photograph the separations or keep the original gels in dryor humid form. However, measurement and further analysis of theelectropherograms is necessary for a number of applications:

. It is difficult, even impossible to determine the intensity of in-dividual fractions visually. For example:Homozygous and heterozygous genotypes must be differen-tiated during genetic investigations. Usually only the presenceor absence of a band or spot can be detected visually.When protein metabolism kinetics is studied, the increase ordecrease of certain fractions must be recognized.

. Single fractions or groups of fractions should be quantified.This is only possible by scanning the separation traces andintegrating the surfaces of the peak diagram with a densit-ometer. It is necessary to measure the zones as exactly as pos-sible since they can present different forms and zone widthsdepending on the method used.

. The interpretation of 2-D electropherograms with several hun-dreds to a few thousand spots is complicated and time-con-suming, which is why computers are essential. The patternsare normally recorded with scanners.

. Densitograms are the usual form of representation in manyareas such as clinical chemistry for example.

. Electrophoresis patterns can often be compared more exactlyby densitometry than by the simple visual comparison.

. Data processing is used more and more often because of theamount of data collected in laboratories. To be able to evaluate,save and process results of electrophoresis by computer, thelanes must be digitalized by a scanner.

. Molecular weights or isoelectric points of samples can beassigned by computer.

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For example measurementsrelative to albumin.

e.g. the identification ofsubstances or simple compar-ison of electrophoresis patterns.

Homozygote means: intenseband. Heterozygote means:partial intensity.

A whole series of factors mustbe taken into account duringquantification. They will bediscussed later.

see 2-D electrophoresis,pages 42 and 92 ff.

In many laboratories, scanningof gels and subsequent printingwith a laser or ink jet printerhas replaced photographing.

5 Interpretation of electropherograms

. For microbiological taxonomy, plant variety determinations,population studies, and clinical genetics lane relationship den-drograms have to be developed.

. For many uses, especially in routine analysis, the separation dis-tances are shortened and the results more difficult to interpretvisually. Bands which lie close to one another can be resolved byhigh resolution scanning and enlarged by the computer.

5.2.1Hardware for image analysis

Prior to the analysis, the electropherograms have to be fed into a com-puter with a video camera, a desktop scanner or a densitometer.

Optical density: A prerequisite for correct and linear values is a suffi-cient light intensity. In high resolution electrophoresis the zones canhave an optical densities (O.D.) over 3 O.D.

The unit O.D. for the optical density is mostly used in biology andbiochemistry and is defined as follows: 1 O.D. is the amount of sub-stance, which has an absorption of 1 when dissolved and measuredin 1 mL in a cuvette with a thickness of 1 cm.

According to Lambert-Beer’s law the extinction of light of a specificwavelength shining through a dissolved substance is proportional toits concentration.

The quantification of results is only possible when the absorptionsare linear. High performance desktop scanners show linear measure-ments up to 3.5 O.D.

Video Cameras: The resolution and sensitivity of video cameras hasbeen considerably improved in the last years. They can be used forvisible and UV light. Video cameras are mostly employed for the eva-luation of relatively small gels, blotting membranes, and Ethidiumbromide stained agarose gels. With cooled CCD cameras very highsensitivity can be achieved. A camera has the advantage, that it canaccumulate signals over a certain time period for detecting weak sig-nals. Modern camera systems are built into light-shielded viewingchambers, no darkrooms are required for fluorescence and chemilu-minescence detection.

Densitometers: Those are mobile photometers which measure elec-trophoresis or thin-layer chromatography lanes. The Rf values andthe extinction (light absorption) of the individual zones are deter-mined. The result is a peak diagram (densitogram). The surfacesunder the peaks can be used for quantification. Such densitometersare still used in routine clinical analysis for the evaluation of celluloseacetate and agarose gels.

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Magnifying glass effect.

The background of blottingmembranes sometimes reaches2.5 O.D.

The intensity of the lightabsorption of a substance iscalled extinction.

CCD cameras provide digitalsignals. They can be coupleddirectly to a computer.

Cameras are mainly used forrelatively small gels up to 15cm separation distance. Forhigh resolution gels, like thoseused for 2-D electrophoresis,scanners are recommended.

Because they are relativelyslow, they are more and morereplaced by scanners.

5.2 Image analysis

Desktop Scanners: High performance instruments are available,which can scan in both, reflectance and transmittance mode, andhave a liquid leakage-free scanning bed (Fig. 43). They scan very fast,provide very high resolution, and they are less expensive than densit-ometers. For quantitative results calibration and scanning in trans-mission mode is a must.

Storage Phosphor Screen Scanners: Autoradiography techniques arethe most sensitive. The detection is much faster with phosphorscreen scanners than with exposures on X-ray films, and they have amuch higher linear dynamic range for quantification. After the expo-sure of a dried gel or a blotting membrane, the storage screen isscanned with a HeNe laser at 633 nm.

Differentially labelled samples can be analysed in the followingway: with direct exposure both 35S and 32P signals are recorded; witha second exposure through a thin copper foil only 32P labelled pro-teins are detected.

Fluorescence Scanners: Staining or labelling proteins with fluorescentdyes show less sensitivity than silver staining, but a much wider lin-ear dynamic range. Modern instruments have a confocal scan head tocancel signals from scattered excitation light, and to reduce fluores-cence background coming from glass plates and other supportingmaterial. The laser light excites the fluorescent label or bound fluor-escent dye, the emitted light of a different wavelength is bundledwith a collection lens and transported to the detector via a fibre opticcable (see Fig. 44). Signals emitted from bands or spots, which areexcited by stray light, are focused out, they will not hit the “peephole”, and will thus not be conducted by the fibre optic cable.

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GelScan XL 24 dec 1990

gel

Fig. 43: Modified desk top scanner with liquidleakage-free scanning bed for rapid high reso-lution scanning of one- and two-dimensionalelectrophoresis gels.

Blotting membranes are mostlyscanned in the reflectancemode.

For reuse, the screen is exposedto extra-bright light to erase theimage.

There is still a drawback forfluorescence detection: the highprice for fluorescence dyescompared to other staining andlabelling methods.

5 Interpretation of electropherograms

Multipurpose Scanners: The functions for storage phosphor imaging,multicolor fluorescence detection, and chemiluminescence can becombined in one instrument. Lasers with different wavelengths arecombined with different filters for the various scanning modes. Thedetectors are usually very sensitive photomultipliers. In Fig. 45 suchan instrument is displayed.

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gel

glass plate

emissionfilter

lens

lens

lens

excitation by

stray light focused beam

laser diode

beamsplitter

fibre opticcable

“peep hole”

tophoto-multiplier

Fig. 44: Schematic diagram of the principle offluorescence detection with confocal optics.

Fig. 45: Variable mode laser scanner. Withkind permission from Amersham PharmaciaBiotech, Sunnyvale, USA.

There is a trend to replaceradioactivity in the laboratorieswherever possible by fluores-cence detection.

5.2 Image analysis

Computer Hardware: Because today’s personal computers have veryfast processors, excellent graphic devices, and can be equipped withhigh capacity hard disks for reasonable prices, it is no longer neces-sary to perform the image analysis on a work station or a UNIX�

based computer. The data can easily be further processed with officesoftwares.

However, there are still two standards existing:

. In numerous academic laboratories Apple Macintosh comput-ers and software are preferred.

. In the industry – but also in many academic laboratories –Windows is used.

Some suppliers provide software for both computer standards.

5.2.2Software for image analysis

Either the evaluation software is linked to the scanning software, oran image can be imported as a grey TIF file for further analysis.

Here are a few general points for what a “good” electrophoresis im-aging software should afford:

. Ease of use, as intuitive as possible.

. Mathematical correct algorithms.

. Fast and automated for high throughput applications.

. Original data must not be changed.

. Editing functions to correct errors.

One-dimensional gel software

Usually an automated 1-D evaluation follows this sequence:

1. lane detection2. correction of distortions (like a smiling front )3. lane correction (if necessary)4. band detection5. band matching6. Rf, Mr, or pI calculation7. background subtraction8. band quantification9. report tables

In Fig. 46 a typical screen of a SDS gel evaluation is shown. Themolecular weights are assigned with the help of marker protein val-ues.

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Personal computers are mucheasier to operate than workstations. The new scanning anddata processing softwares arevery convenient and easy tohandle.

Color scans are impossible toevaluate.

A manual should not beneeded

Reproducible calculations

Evaluation is often a bottleneck

Except permissible adjustments

With reporting

Additional functions allow:– lane matching,– normalization for the densitycomparison between differentgels, and– data base methods like– clustering for dendrogramconstructions and– sample identification.

5 Interpretation of electropherograms88

Fig. 46: Computer screen showing the detected lanes of an SDS gel and thedensitogram of a selected lane with annotated molecular weights.

Fig. 47: Computer screen of 2-D electrophoresis image analysis software. In thebackground: 2-D images with automatically detected spots; in the foreground:volume table, histograms and three-dimensional representation of selectedspots.

5.2 Image analysis

Two-dimensional gel software

For automated 2-D evaluation the following path is taken:

1. spot detection and spot filtering2. background correction3. spot volume calculation4. 2-D calibration5. averaging multiple gels of a sample6. choose reference gel7. spot matching8. normalization9. pattern comparison10. statistical evaluation

Fig. 47 displays the computer screen of digitized and processed im-ages of 2-D electrophoresis gels. The samples were extracts from dif-ferent bacterial cultures grown on different sugar media. The volumetables and histograms of spot intensities represent the quantitativecontents of selected bacterial proteins grown on the different media.The three-dimensional representation is particularly useful for thedetection of low expressed proteins next to highly abundant proteins.

Particularly for 2-D gels the use of quick and automated evaluationsoftware is important, because it is impossible to find pattern differ-ences with the eye. Sophisticated database functions are required tosearch across different images and experiments.

Critical issues in quantification

a) Ratio absorption : concentration

It should first be understood that the conventional laws of photome-try do not apply to densitometry. While dilute solutions (mmol con-centrations) are measured in the UV and visible ranges during photo-metry, the absorbing medium is a highly concentrated precipitate in

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peak area200

150

100

50

00 200 400 600 800 1,000

protein amount in µg

Fig. 48: Color intensity curve of carbonicanhydrase, separated with SDS-PAGE andstained with colloidal Coomassie BrilliantBlue G-250.

There is a major difference to1-D evaluation: One gelcorresponds to one sample.

It is very complicated, if notimpossible, to calculate aconstant for a protein-dyeaggregate.

5 Interpretation of electropherograms

gel densitometry. It usually is a protein which is bound to a chromo-phore. A typical protein curve is shown in Fig. 48. Similar problemsoccur during densitometric evaluation of X-ray films. The Lambert-Beer law cannot be applied to densitometry because it is only valid forvery dilute, “ideal” sample solutions.

b) External standard

An external standard should be used for a series of experiments. If amixture of proteins is used, the amount of each standard proteinapplied is known. If the amount of a specific protein is to be calcu-lated in mg, it should be remembered that every protein has a differ-ent affinity for a dye, e.g. Coomassie (Fig. 49).

The diagram is based on the calculation of the amount of marker

protein in the mixture per measured and integrated peak area:

Lactalbumin 138 lg Carbonic anhydrase (CA) 108 lgTrypsin inhibitor 133 lg Phosphorylase B 60 lgOvalbumin 122 lg BSA 57 lg

Therefore an unknown protein should not be calculated directlyusing albumin for calibration, the protein itself should be available inthe pure form. But if the values are recorded compared to albumin,the correct values can be recalculated later.

The path to an absolute value lies through the identification of aprotein with immuno or lectin blotting and the use of the proceduredescribed above. If there is no antibody or specific ligand available, asubject for a thesis has been found!

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Lactalbumin TI Ovalbumin CA Phosphorylase BSA

160

140

120

20

40

60

80

100

0

amount of protein ( g / peak area)�

Fig 49: Diagram of the protein amount ofindividual markers per integrated peak area.TI: trypsin inhibitor from soybeans. Stainingwith Coomassie Brilliant Blue G-250.

Recalculated means: if theprotein to be determinedshould later become availableas pure substance, a compar-ison can then be carried out.