A scientific life with chemistry, optics, and mathematics

Rilbe, Harry

doi:10.1002/elps.1150050102

Cited by 7 publications

(6 citation statements)

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“…Vesterberg found suitable carrier ampholytes for various parts of the pH scale [23]. The beginning of IEF, with the concept of ampholytes, and its further development was well documented by Rilbe himself [24,25] in 1976 and 1984 in two papers in which he, in great detail and in a pleasing historic manner, describes the birth of ampholytes. The work of Rilbe and Vesterberg, and also the development of synthetic polyacrylamide gel by Raymond and Weintraub [26], led to numerous publications dealing with applications of IEF in plant sciences.…”

Section: The Concept Of Carrier Ampholytesmentioning

confidence: 97%

Isoelectric focusing in the vegetable seed industry

Berg¹

1998

Electrophoresis

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Different types of electrophoretic procedures were developed for hybrid purity testing based on starch gel electrophoresis (SGE), vertical polyacrylamide gel electrophoresis (PAGE), and isoelectric focusing (IEF). For the most important vegetables these methods are much faster than plant grow-outs and relatively inexpensive. Compared to SGE and PAGE methods, horizontal IEF proved to be more efficient for large-scale hybrid purity testing. These developments were made possible by the basic work of Harry Rilbe and improvements that were initiated as a result of Rilbe's work. The present paper describes a number of milestones during this developmental period, starting with the isoelectric focusing concept of Harry Rilbe up to the large-scale application of IEF. Further, a comparison of IEF with DNA fingerprinting methods along with the future of both techniques is discussed with respect to hybrid purity testing in the vegetable seed industry. When it comes to a choice between the use of either IEF or a DNA-based method, efficiency and efficacy determines the method which is best suited for hybrid purity testing. It is also concluded that in the future we will see an increased use of both IEF as well as DNA-based methods for hybrid purity testing because expectations of growers has increased; consequently they will accept fewer inbreds in a hybrid variety, especially when growing in a greenhouse.

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Section: The Concept Of Carrier Ampholytesmentioning

confidence: 97%

Isoelectric focusing in the vegetable seed industry

Berg¹

1998

Electrophoresis

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“…[95–106] in Ref. [5], along with the periodic Recent Progress in IEF reviews in Electrophoresis ). The notion that Svensson's IEF experiment constitutes a steady‐state separation method has been universally accepted and became part of the codified core of modern separation science (see, e.g., Giddings’ classification of IEF as an Sc method, a “Static, continuous field, steady‐state” separation method, Chapters 7.3 and 8.10 in Ref.…”

Section: Evolution Of the Ief Experiments Viewed Through Today's Hind...mentioning

confidence: 99%

“…With both his apparatus [3] and Vesterberg's CA mixtures [4] becoming commercially available, IEF revolutionized electrophoretic protein separations (see Rilbe's recollections in Ref. [5], the large number of textbooks on IEF, e.g., Refs. [6,7], and the proceedings volumes of international IEF symposia, Refs.…”

Section: Evolution Of the Ief Experiments Viewed Through Today's Hind...mentioning

confidence: 99%

Evolution of the theoretical description of the isoelectric focusing experiment: I. The path from Svensson's steady‐state model to the current two‐stage model of isoelectric focusing

Vigh

Gaš

2023

Electrophoresis

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In 1961, Svensson described isoelectric focusing (IEF), the separation of ampholytic compounds in a stationary, natural pH gradient that was formed by passing current through a sucrose density gradient‐stabilized ampholyte mixture in a constant cross‐section apparatus, free of mixing. Stable pH gradients were formed as the electrophoretic transport built up a series of isoelectric ampholyte zones—the concentration of which decreased with their distance from the electrodes—and a diffusive flux which balanced the generating electrophoretic flux. When polyacrylamide gel replaced the sucrose density gradient as the stabilizing medium, the spatial and temporal stability of Svensson's pH gradient became lost, igniting a search for the explanation and mitigation of the loss. Over time, through a series of insightful suggestions, the currently held notion emerged that in the modern IEF experiment—where the carrier ampholyte (CA) mixture is placed between the anolyte‐ and catholyte‐containing large‐volume electrode vessels (open‐system IEF)—a two‐stage process operates that comprises a rapid first phase during which a linear pH gradient develops, and a subsequent slow, second stage, during which the pH gradient decays as isotachophoretic processes move the extreme pI CAs into the electrode vessels. Here we trace the development of the two‐stage IEF model using quotes from the original publications and point out critical results that the IEF community should have embraced but missed. This manuscript sets the foundation for the companion papers, Parts 2 and 3, in which an alternative model, transient bidirectional isotachophoresis is presented to describe the open‐system IEF experiment.

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“…Once all these conditions are fulfilled, adjacent, individual carrier ampholyte bands form across the separation compartment such that the pH in the successive bands increases monotonously from that in the anolyte to that in the catholyte. If the concentration of the carrier ampholyte in its band is sufficiently high, the pH approaches the isoprotic/isoelectric value of the individual carrier ampholyte such that, to quote Rilbe , “… my contemplated pH gradients should be stable indefinitely.”.…”

Section: Introductionmentioning

confidence: 99%

CE determination of the thermodynamic pK_a values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes‐based IEF and its numeric simulation

et al. 2019

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CE determination of the thermodynamic pK a values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes-based IEF and its numeric simulation Fourteen low molecular mass UV absorbing ampholytes containing 1 or 2 weakly acidic and 1 or 2 weakly basic functional groups that best satisfy Rilbe's requirement for being good carrier ampholytes ( pK a = pKa monoanion -pKa monocation < 2) were selected from a large group of commercially readily available ampholytes in a computational study using two software packages (ChemSketch and SPARC). Their electrophoretic mobilities were measured in 10 mM ionic strength BGEs covering the 2 < pH < 12 range. Using our Debye-Hückel and Onsager-Fuoss laws-based new software, AnglerFish (freeware, https://echmet.natur.cuni.cz/software/download), the effective mobilities were recalculated to zero ionic strength from which the thermodynamic pK a values and limiting ionic mobilities of the ampholytes were directly calculated by Henderson-Hasselbalch equation-type nonlinear regression. The tabulated thermodynamic pK a values and limiting ionic mobilities of these ampholytes (pI markers) facilitate both the overall and the narrow-segment characterization of the pH gradients obtained in IEF in order to mitigate the errors of analyte ampholyte pI assignments caused by the usual (but rarely proven) assumption of pH gradient linearity. These thermodynamic pK a and limiting mobility values also enable the reality-based numeric simulation of the IEF process using, for example, Simul (freeware, https://echmet.natur.cuni.cz/software/download). Abbreviations:3MHIS, 3-methylhistidine; DNS-Cl, dansylchloride; DNS-IDA, dansylated iminodiacetic acid; GLYHIS, glycyl-histidine; ICIEF, Imaging capillary isoelectric focusing; IDA, iminodiacetic acid; LAB, labetalol; LBB, Leucoberbelin blue I dye; SERO, serotonin compartment has to be filled with a base (the catholyte, e.g. 20 mM NaOH). Fourth, convective disturbances have to be absent or suppressed in the system (either by using an anti-convective medium or a capillary as the separation compartment). Fifth, a sufficiently large DC voltage has to be applied between the anode and the cathode for a sufficiently long period of time to allow any and all of the initially cationic and anionic carrier ampholytes in the mixture to migrate out of sections of the separation compartment in which the local pH value is different from their isoelectric point value. (Since Rilbe considered the isoelectric point value to be very close to the isoprotic point value-which he calculated as (pKa monocation + pKa monoanion )/2-the symbol pI, and the term pI value, became loosely-and interchangeably-used in the literature to designate either "iso" value.)Once all these conditions are fulfilled, adjacent, individual carrier ampholyte bands form across the separation compartment such that the pH in the successive bands increases Color online: See the article online to view Figs. 1, 3-5 in ...

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A scientific life with chemistry, optics, and mathematics

Cited by 7 publications

References 66 publications

Isoelectric focusing in the vegetable seed industry

Isoelectric focusing in the vegetable seed industry

Evolution of the theoretical description of the isoelectric focusing experiment: I. The path from Svensson's steady‐state model to the current two‐stage model of isoelectric focusing

CE determination of the thermodynamic pK_a values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes‐based IEF and its numeric simulation

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A scientific life with chemistry, optics, and mathematics

Cited by 7 publications

References 66 publications

Isoelectric focusing in the vegetable seed industry

Isoelectric focusing in the vegetable seed industry

Evolution of the theoretical description of the isoelectric focusing experiment: I. The path from Svensson's steady‐state model to the current two‐stage model of isoelectric focusing

CE determination of the thermodynamic pKa values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes‐based IEF and its numeric simulation

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CE determination of the thermodynamic pK_a values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes‐based IEF and its numeric simulation