226:
vessels with a capillary passing through each vessel. Part of the capillary in each vessel is replaced by a semipermeable membrane. The vessels contain buffer solutions with different pH values, so that a pH gradient is effectively established inside the capillary. The buffer solution in each vessel has an electrical contact with a voltage divider connected to a high-voltage power supply, which establishes an electrical field along the capillary. When a sample (a mixture of peptides or proteins) is injected in the capillary, the presence of the electrical field and the pH gradient separates these molecules according to their isoelectric points. The multi-junction IEF system has been used to separate tryptic peptide mixtures for two-dimensional proteomics and blood plasma proteins from
105:(pI) will be positively charged and so will migrate toward the cathode (negatively charged electrode). As it migrates through a gradient of increasing pH, however, the protein's overall charge will decrease until the protein reaches the pH region that corresponds to its pI. At this point it has no net charge and so migration ceases (as there is no electrical attraction toward either electrode). As a result, the proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI. The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands.
31:
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128:
which the pH of that molecule's isoelectric point is reached. At this point the molecule no longer has a net electric charge (due to the protonation or deprotonation of the associated functional groups) and as such will not proceed any further within the gel. The gradient is established before adding the particles of interest by first subjecting a solution of small molecules such as
853:
127:
end. Negatively charged molecules migrate through the pH gradient in the medium toward the "positive" end while positively charged molecules move toward the "negative" end. As a particle moves toward the pole opposite of its charge it moves through the changing pH gradient until it reaches a point in
225:
The increased demand for faster and easy-to-use protein separation tools has accelerated the evolution of IEF towards in-solution separations. In this context, a multi-junction IEF system was developed to perform fast and gel-free IEF separations. The multi-junction IEF system utilizes a series of
199:
cells perform isoelectric focusing of proteins in their interior to overcome a limitation of the rate of metabolic reaction by diffusion of enzymes and their reactants, and to regulate the rate of particular biochemical processes. By concentrating the enzymes of particular metabolic pathways into
166:
where a pH gradient has been established. Gels with large pores are usually used in this process to eliminate any "sieving" effects, or artifacts in the pI caused by differing migration rates for proteins of differing sizes. Isoelectric focusing can resolve proteins that differ in
247:
Bjellqvist, Bengt; Ek, Kristina; Righetti, Pier
Giorgio; Gianazza, Elisabetta; Görg, Angelika; Westermeier, Reiner; Postel, Wilhelm (1982). "Isoelectric focusing in immobilized pH gradients: Principle, methodology and some applications".
216:
since it has the potential to provide rapid protein analysis, straightforward integration with other microfluidic unit operations, whole channel detection, nitrocellulose films, smaller sample sizes and lower fabrication costs.
200:
distinct and small regions of its interior, the cell can increase the rate of particular biochemical pathways by several orders of magnitude. By modification of the isoelectric point (pI) of molecules of an enzyme by, e.g.,
93:
gel matrix co-polymerized with the pH gradient, which result in completely stable gradients except the most alkaline (>12) pH values. The immobilized pH gradient is obtained by the continuous change in the ratio of
573:
Pirmoradian, M.; Zhang, B.; Chingin, K.; Astorga-Wells, J.; Zubarev R.A. (2014). "Membrane-assisted isoelectric focusing device as a micro-preparative fractionator for two dimensional shotgun proteomics".
525:"Multijunction Capillary Isoelectric Focusing Device Combined with Online Membrane-Assisted Buffer Exchanger Enables Isoelectric Point Fractionation of Intact Human Plasma Proteins for Biomarker Discovery"
376:
Kastenholz, B (2004). "Preparative Native
Continuous Polyacrylamide Gel Electrophoresis (PNC-PAGE): An Efficient Method for Isolating Cadmium Cofactors in Biological Systems".
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or dephosphorylation, the cell can transfer molecules of the enzyme between different parts of its interior, to switch on or switch off particular biochemical processes.
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that takes advantage of the fact that overall charge on the molecule of interest is a function of the
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Molecules to be focused are distributed over a medium that has a pH gradient (usually created by
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Baskin E.F.; Bukshpan S; Zilberstein G V (2006). "pH-induced intracellular protein transport".
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Stryer, Lubert: "Biochemie", page 50. Spektrum
Akademischer Verlag, 1996 (German)
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Scheme of isoelectric focusing with immobilized pH gradient (IPG) gels.
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value by as little as 0.01. Isoelectric focusing is the first step in
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98:. An immobiline is a weak acid or base defined by its pK value.
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183:. Isoelectric focusing, on the other hand, is the only step in
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Microchip based electrophoresis is a promising alternative to
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330:. Methods in Enzymology. Vol. 182. pp. 459–77.
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Isoelectric
Focusing: Theory, Methodology and Application
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The method is applied particularly often in the study of
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Pirmoradian M.; Astorga-Wells, J.; Zubarev, RA. (2015).
139:, which separate based on their relative content of
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is passed through the medium, creating a "positive"
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250:Journal of Biochemical and Biophysical Methods
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421:"Does a cell perform isoelectric focusing?"
132:with varying pI values to electrophoresis.
101:A protein that is in a pH region below its
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49:, is a technique for separating different
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7:
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780:Gel electrophoresis of nucleic acids
709:Electrophoretic mobility shift assay
775:DNA separation by silica adsorption
754:Two-dimensional gel electrophoresis
195:According to some opinions, living
173:two-dimensional gel electrophoresis
739:Polyacrylamide gel electrophoresis
384:(4). Informa UK Limited: 657–665.
230:patients for biomarker discovery.
25:
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840:
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744:Pulsed-field gel electrophoresis
324:(1990). "Isoelectric focusing".
785:Gel electrophoresis of proteins
734:Moving-boundary electrophoresis
674:Capillary electrochromatography
689:Difference gel electrophoresis
1:
790:Serum protein electrophoresis
694:Discontinuous electrophoresis
327:Guide to Protein Purification
902:Molecular biology techniques
544:10.1021/acs.analchem.5b03344
448:10.1016/0303-2647(90)90005-L
336:10.1016/0076-6879(90)82037-3
262:10.1016/0165-022X(82)90013-6
669:Agarose gel electrophoresis
57:(pI). It is a type of zone
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648:History of electrophoresis
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827:Electrophoresis (journal)
679:Capillary electrophoresis
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491:10.1088/1478-3975/3/2/002
214:capillary electrophoresis
89:(IPG) gels. IPGs are the
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53:by differences in their
208:Microfluidic chip based
185:preparative native PAGE
152:immobilized pH gradient
87:immobilized pH gradient
81:IEF involves adding an
27:Type of electrophoresis
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719:Immunoelectrophoresis
704:Electrochromatography
292:Pier Giorgio Righetti
73:of its surroundings.
61:usually performed on
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892:Industrial processes
865:Analytical Chemistry
811:Isoelectric focusing
576:Analytical Chemistry
532:Analytical Chemistry
390:10.1081/al-120029742
39:Isoelectric focusing
806:Electrical mobility
714:Gel electrophoresis
538:(23): 11840–11846.
483:2006PhBio...3..101B
440:1990BiSys..24..127F
322:David Edward Garfin
228:Alzheimer's disease
378:Analytical Letters
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103:isoelectric point
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657:Techniques
428:BioSystems
234:References
197:eukaryotic
113:ampholytes
91:acrylamide
398:0003-2719
270:0165-022X
110:aliphatic
83:ampholyte
77:Procedure
51:molecules
846:Category
820:Journals
596:24824042
560:26531800
507:41599078
499:16829696
406:97636537
181:SDS-PAGE
179:through
148:residues
137:proteins
63:proteins
858:Commons
479:Bibcode
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436:Bibcode
354:2314254
278:7142660
164:agarose
125:cathode
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162:, or
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