A tribute to Angelika Görg

4836 Proteomics 2008, 8, 4836–4839

A tribute to Angelika Görg

From noodles to solid science: How did it come to our

using IPG strips?

Although the idea had been applied before [1], the first impressive example ofhigh resolution 2-D electrophoresis, coupling denaturing isoelectric focusing (IEF)to SDS electrophoresis, was published in 1975 [2]. At that time where DNA clon-ing and sequencing were in their infancy, and where protein biochemistry wasproportionally further developed than it is now, these impressive results promptedmany researchers to use this technique. However, most (including me) discoveredthat this technique was more an art than a science, and was quite delicate tomaster. First of all, it must be recalled that IEF is quite sensitive to many chemi-cals, including salts. Thus, when different samples are loaded on an IEF gel plate,it is fairly common that the electric field is not homogeneous, which results indistorted migrations, and thus in comma-shaped migration lanes. These are ofcourse impossible to transfer to a second dimension gel. Thus, at that time IEFgels for 2-D electrophoresis were tube gels, of small diameter to facilitate transferonto the second dimension gel. Furthermore, as a good IEF separation impliesnot to induce molecular sieving of the proteins in the gel, the IEF gels are alwayslow percentage gels (4-5% acrylamide). Thus, a typical IEF gel was very much likea rice noodle: transparent and soft. Extruding it from its gel tube, where it had toadhere firmly to avoid migration artefacts, to the equilibration solution, was moresport than science. It was not uncommon to induce variable stretching of the tubegels, so that gels cast at an equal length of 16 cm could measure anything be-tween 16 and 20 cm, of course with a non-linear deformation. Then, fishing theIEF gel from its equilibration solution and putting it in place (without too muchstretching please!) on top of the second dimension gel plate required a consider-able amount of dexterity.

In addition to these mechanical adventures, classical IEF with carrier ampho-lytes was (and still is) full of caveats. The first one is due to the way ampholytesare made, i.e. by bulk reaction of unsaturated acidic compounds onto a poly-amine, or by polymerization of amines and amino acids with epichlorhydrin.From a chemical point of view, the result is a terrible brew, but it does what it isasked to do, i.e. stabilize a pH gradient. However, between two different batches,the real composition of the brew is different, so that the resulting pH gradient isalso different. This of course poses real reproducibility problems, so some labsbought a whole ampholyte batch, aliquoted it and then stored it in a deep-freezeto avoid this issue. Last but by no means least, carrier ampholyte-based IEF hasan intrinsic weakness, i.e. cathodic drift. This means that the pH gradient is notstable over time, and crumbles at the basic end, with two obvious consequences:

i) migration had to be carefully controlled for it to be reproducibleii) basic proteins were impossible to analyze with the classical IEF techniques,

and required the development of even more difficult-to-master techniques, namelynon-equilibrium pH gradient gel electrophoresis [3].

TRIBUTE

Thierry Rabilloud

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2008, 8, 4836–4839 4837

Thus, although the resolution was available, making a complete series of re-producible 2-D gels required quite some effort and dedication. These problems ofpH gradient stability induced much research at the end of the 70s – more detailscan be found in an excellent book by P. G Righetti [4] – and the answer finallycame with immobilized pH gradients [5]. To be honest, it was just the beginningof the answer. While the use of grafted buffering chemicals copolymerized in theacrylamide gel was theoretically perfect, it was hard to make it work. As a matterof fact, making an immobilized pH gradient, and especially one which is severalpH units wide, is like trying to make a uniform copolymerization of several dif-ferent monomers, and causes a lot of problems: how to make the polymerizationuniform, how to remove the unreacted monomers which would otherwise ruinyour pH gradient, how to infuse them in the washed gel the chemicals need todenature the proteins etc...

It took years for dedicated researchers, gathered around P. G. Righetti, tobring the idea of immobilized pH gradients to functional IEF in gel plates. At thattime, Angelika Görg had already entered on the scene as an expert in plasticsupported thin acrylamide gels [6]. But other challenges were to be met by thecommunity, namely how to interface an immobilized pH gel with a second di-mension gel. Some tried to use tube gels, so popular at that time [7, 8], but thetube gels could not be washed to eliminate unpolymerized monomers, and mak-ing this work led to loss of most of the immobilized pH gradients advantages.Others [9] used a washed IPG plate, in which the gel was scraped in some placesto delineate gel lanes on which the sample was loaded. This required a steadyhand and a long knife, and to be honest, this was not much more practicable thangel tubes.

At that time, the nice reswelling properties of IPG gels [10] were alreadyempirically known. Thus, the key idea of Angelika Görg was to cut the gel platewhile dry (which is much easier than when wet), to reswell it in the appropriaterehydration buffer, and to load the sample on it. Everyone had predicted terribleside effects on such a narrow gel lane, but this proved to work with impeccableprotein banding and impeccable resulting 2D gels [11, 12]. IPG strips were born,and it was soon demonstrated how reproducible they were, without any cathodicdrift and with perfect reproducibility not to mention the added advantages of easyhandling and the absence of operator-induced gel deformations.

This was a major contribution to the field, as it switched 2-D electrophoresisfrom a difficult art to good craftsmanship, as is all protein biochemistry. But thestory is not over. Ampholytes gels behave nicely when interfaced on a SDS gel.They are indeed plain acrylamide gels, containing ampholytes that migrate nicelywith the SDS front. IPG gels are not the same: containing charge monomers, theyare keen to migrate as a whole in the electric field, and thus to dig their way intothe second dimension gel, with collateral problems in protein transfer betweenboth gels. Here again, Angelika made a major contribution by introducing thedouble equilibration in the presence of SDS, urea and glycerol, and DTT in thefirst bath, followed by iodoacetamide in the second bath [13]. Everyone does itnowadays, it has become lab routine, but not everyone quotes this second seminalpaper.


4838 Proteomics 2008, 8, 4836–4839

With this chain of three key contributions ((i) getting the adequate chemicals,(ii) making it work in IEF and (iii) making it work in 2-D gels), made by threedifferent research groups, the ground were set for the democratization of 2-Dgels. Other adventures were to come, such as exploring the focusing of very basicproteins [14, 15], but this is another part of the story.

Many people opening a pack of strips and making their routine 2-D gels withwhat they think to be standard protocols do not imagine the amount of sweat,time, effort, dedication that was needed to make this such a simple process (and Ido not count sparks and gel burnings!). However, this is a real tribute we all oweto only a few people, among whom Angelika Görg is a major player.

Thierry RabilloudPROTEOMICS Senior Editor

References

[1] MacGillivray, A. J., Rickwood, D., The heterogeneity of mouse-chromatin nonhistoneproteins as evidenced by two-dimensional polyacrylamide-gel electrophoresis andion-exchange chromatography. Eur. J. Biochem. 1974, 41, 181–190.

[2] O’Farrell, P. H., High resolution two-dimensional electrophoresis of proteins. J. Biol.Chem. 1975, 250, 4007–4021.

[3] O’Farrell, P. Z., Goodman, H. M., O’Farrell, P. H., High resolution two-dimensionalelectrophoresis of basic as well as acidic proteins. Cell 1977, 12, 1133–1141.

[4] Righetti, P. G., Isoelectric Focusing: Theory, Methodology and Applications. Elsevier,Amsterdam 1983.

[5] Bjellqvist, B., Ek, K., Righetti, P. G., Gianazza, E. et al., Isoelectric focusing in immobi-lized pH gradients: principle, methodology and some applications. J. Biochem. Bio-phys. Methods 1982, 6, 317–339.

[6] Görg, A., Postel, W., Westermeier, R., Gianazza, E., Righetti, P. G., Gel gradient elec-trophoresis, isoelectric focusing and two-dimensional techniques in horizontal,ultrathin polyacrylamide layers. J. Biochem. Biophys. Methods 1980, 3, 273–284.

[7] Hochstrasser, D., Augsburger, V., Funk, M., Appel, R. et al., Immobilized pH gradientsin capillary tubes and two-dimensional gel electrophoresis. Electrophoresis 1986, 7,505–511.

[8] Asakawa, J. I., Two-dimensional gel electrophoresis of platelet polypeptides withimmobilized pH gradients in capillary tubes. Electrophoresis 1988, 9, 562–568.

[9] Gianazza, E., Astrua-Testori, S., Giacon, P., Righetti, P. G., An improved protocol fortwo-dimensional maps of serum proteins with immobilized pH gradients in the firstdimension. Electrophoresis 1985, 6, 332–339.

[10] Righetti, P. G., Of matrices and men. J. Biochem. Biophys. Methods 1989, 19, 1–20.


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[11] Strahler, J. R., Hanash, S. M., Somerlot, L., Weser, J. et al., High resolution two-dimensional polyacrylamide gel electrophoresis of basic myeloid polypeptides: Useof immobilized pH gradients in the first dimension. Electrophoresis 1987, 8, 165–173.

[12] Görg, A., Postel, W., Günther, S., Weser, J. et al., Approach to stationary two-dimen-sional pattern: Influence of focusing time and immobiline/carrier ampholytes con-centrations. Electrophoresis 1988, 9, 37–46.

[13] Görg, A., Postel, W., Weser, J., Günther, S. et al., Elimination of point streaking on sil-ver stained two-dimensional gels by addition of iodoacetamide to the equilibrationbuffer. Electrophoresis 1988, 9, 122–124.

[14] Righetti, P. G., Bossi, A., Görg, A., Obermaier, C., Boguth, G., Steady-state two-dimensional maps of very alkaline proteins in an immobilized pH 10-12 gradient, asexemplified by histone types. J. Biochem. Biophys. Methods 1996, 31, 81–91.

[15] Görg, A., Obermaier, C., Boguth, G., Csordas, A. et al., Very alkaline immobilized pHgradients for two-dimensional electrophoresis of ribosomal and nuclear proteins.Electrophoresis 1997, 18, 328–337.


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A tribute to Angelika Görg