215:
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
94:(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.
20:
830:
842:
117:
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
116:
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
214:
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
188:
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
155:
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
236:
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".
205:
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.
189:
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.,
82:
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
562:
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".
514:"Multijunction Capillary Isoelectric Focusing Device Combined with Online Membrane-Assisted Buffer Exchanger Enables Isoelectric Point Fractionation of Intact Human Plasma Proteins for Biomarker Discovery"
365:
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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164:, in which proteins are first separated by their pI value and then further separated by
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Stryer, Lubert: "Biochemie", page 50. Spektrum
Akademischer Verlag, 1996 (German)
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139:, whose value is represented by the pI. Proteins are introduced into an
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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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39:
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87:. An immobiline is a weak acid or base defined by its pK value.
172:. Isoelectric focusing, on the other hand, is the only step in
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129:
201:
Microchip based electrophoresis is a promising alternative to
109:
235:
319:. Methods in Enzymology. Vol. 182. pp. 459–77.
286:
Isoelectric
Focusing: Theory, Methodology and Application
124:
The method is applied particularly often in the study of
512:
Pirmoradian M.; Astorga-Wells, J.; Zubarev, RA. (2015).
59:
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128:, which separate based on their relative content of
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is passed through the medium, creating a "positive"
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229:
507:
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239:Journal of Biochemical and Biophysical Methods
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358:
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410:"Does a cell perform isoelectric focusing?"
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121:with varying pI values to electrophoresis.
90:A protein that is in a pH region below its
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603:
364:
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38:, is a technique for separating different
738:Temperature gradient gel electrophoresis
18:
407:
868:
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841:
769:Gel electrophoresis of nucleic acids
698:Electrophoretic mobility shift assay
764:DNA separation by silica adsorption
743:Two-dimensional gel electrophoresis
624:
184:According to some opinions, living
162:two-dimensional gel electrophoresis
13:
728:Polyacrylamide gel electrophoresis
373:(4). Informa UK Limited: 657–665.
219:patients for biomarker discovery.
14:
902:
209:
840:
829:
828:
733:Pulsed-field gel electrophoresis
313:(1990). "Isoelectric focusing".
774:Gel electrophoresis of proteins
723:Moving-boundary electrophoresis
663:Capillary electrochromatography
179:
678:Difference gel electrophoresis
555:
451:
401:
349:
303:
1:
779:Serum protein electrophoresis
683:Discontinuous electrophoresis
316:Guide to Protein Purification
222:
891:Molecular biology techniques
533:10.1021/acs.analchem.5b03344
437:10.1016/0303-2647(90)90005-L
325:10.1016/0076-6879(90)82037-3
251:10.1016/0165-022X(82)90013-6
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658:Agarose gel electrophoresis
46:(pI). It is a type of zone
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637:History of electrophoresis
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816:Electrophoresis (journal)
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668:Capillary electrophoresis
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480:10.1088/1478-3975/3/2/002
203:capillary electrophoresis
78:(IPG) gels. IPGs are the
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42:by differences in their
197:Microfluidic chip based
174:preparative native PAGE
141:immobilized pH gradient
76:immobilized pH gradient
70:IEF involves adding an
16:Type of electrophoresis
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708:Immunoelectrophoresis
693:Electrochromatography
281:Pier Giorgio Righetti
62:of its surroundings.
50:usually performed on
22:
881:Industrial processes
854:Analytical Chemistry
800:Isoelectric focusing
565:Analytical Chemistry
521:Analytical Chemistry
379:10.1081/al-120029742
28:Isoelectric focusing
795:Electrical mobility
703:Gel electrophoresis
527:(23): 11840–11846.
472:2006PhBio...3..101B
429:1990BiSys..24..127F
311:David Edward Garfin
217:Alzheimer's disease
367:Analytical Letters
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673:Dielectrophoresis
577:10.1021/ac404180e
571:(12): 5728–5732.
296:978-0-08-085880-7
92:isoelectric point
44:isoelectric point
34:), also known as
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283:(1 April 2000).
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145:polyacrylamide
119:polyampholytes
74:solution into
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180:Living cells
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85:immobilines
870:Categories
646:Techniques
417:BioSystems
223:References
186:eukaryotic
102:ampholytes
80:acrylamide
387:0003-2719
259:0165-022X
99:aliphatic
72:ampholyte
66:Procedure
40:molecules
835:Category
809:Journals
585:24824042
549:26531800
496:41599078
488:16829696
395:97636537
170:SDS-PAGE
168:through
137:residues
126:proteins
52:proteins
847:Commons
468:Bibcode
445:2249006
425:Bibcode
343:2314254
267:7142660
153:agarose
114:cathode
788:Theory
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130:acidic
104:). An
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413:(PDF)
391:S2CID
151:, or
134:basic
110:anode
54:in a
581:PMID
545:PMID
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441:PMID
383:ISSN
339:PMID
329:ISBN
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263:PMID
255:ISSN
132:and
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321:doi
247:doi
56:gel
32:IEF
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158:pI
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Text is available under the Creative Commons Attribution-ShareAlike License. Additional terms may apply.
