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Scheme of Isoelectric focusing

Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis, usually performed on proteins in a gel, that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.

Isoelectric focusing in laboratory[edit source | edit]

IEF involves adding an ampholyte solution into immobilized pH gradient (IPG) gels. IPGs are the acrylamide 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 Immobilines. An Immobiline is a weak acid or base defined by its pK value.

A protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will migrate towards the cathode (negative). 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 towards 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.

Molecules to be focused are distributed over a medium that has a pH gradient (usually created by aliphatic ampholytes). An electric current is passed through the medium, creating a "positive" anode and "negative" cathode 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 towards the pole opposite of its charge it moves through the changing pH gradient until it reaches a point in which the pH of that molecules 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 polyampholytes with varying pI values to electrophoresis.

The method is applied particularly often in the study of proteins, which separate based on their relative content of acidic and basic residues, whose value is represented by the pI. Proteins are introduced into an Immobilized pH gradient gel composed of polyacrylamide, starch, or agarose 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 pI value by as little as 0.01.^[1] Isoelectric focusing is the first step in two-dimensional gel electrophoresis, in which proteins are first separated by their pI and then further separated by molecular weight through SDS-PAGE.

Isoelectric focusing by living cells[edit source | edit]

According to some opinions,^[2]^[3] living eukaryotic 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 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., phosphorylation 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.

Microfluidic chip based isoelectric focusing[edit source | edit]

Microchip based electrophoresis is a promising alternative to capillary electrophoresis 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.

References[edit source | edit]

^ Stryer, Lubert: "Biochemie", page 50. Spektrum Akademischer Verlag, 1996 (German)
^ Flegr J (1990). "Does a cell perform isoelectric focusing?". BioSystems 24 (2): 127–133. doi:10.1016/0303-2647(90)90005-L. PMID 2249006.
^ Baskin E.F., Bukshpan S, Zilberstein G V (2006). "pH-induced intracellular protein transport". Physical Biology 3 (2): 101–106. doi:10.1088/1478-3975/3/2/002. PMID 16829696.

English Journal

Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis.

Choi H1, Choi N2, Lim B1, Kim TW1, Song S3, Kim YP4.
Biosensors & bioelectronics.Biosens Bioelectron.2015 Nov 15;73:93-9. doi: 10.1016/j.bios.2015.05.047. Epub 2015 May 25.
We report a simple method for analyzing sequential phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic isoelectric focusing (μIEF) electrophoresis. When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-a
PMID 26050965

Capillary isoelectric focusing method development and validation for investigation of recombinant therapeutic monoclonal antibody.

Suba D1, Urbányi Z2, Salgó A3.
Journal of pharmaceutical and biomedical analysis.J Pharm Biomed Anal.2015 Oct 10;114:53-61. doi: 10.1016/j.jpba.2015.04.037. Epub 2015 May 6.
Capillary isoelectric focusing (cIEF) is a basic and highly accurate routine analytical tool to prove identity of protein drugs in quality control (QC) and release tests in biopharmaceutical industries. However there are some "out-of-the-box" applications commercially available which provide easy an
PMID 26025812

Fetal akinesia deformation sequence due to a congenital disorder of glycosylation.

Ganetzky R1,2, Izumi K1,2, Edmondson A3, Muraresku CC2, Zackai E1,3, Deardorff M1,3, Ganesh J4.
American journal of medical genetics. Part A.Am J Med Genet A.2015 Oct;167(10):2411-7. doi: 10.1002/ajmg.a.37184. Epub 2015 May 31.
Congenital disorders of Glycosylation (CDG) are increasingly emerging as a major underlying etiology for patients with complex neurogenetic malformations and dysmorphic features. We describe a newborn female with arthrogryposis multiplex due to fetal akinesia secondary to CDG-DPAGT1. Pregnancy was c
PMID 26033833

Japanese Journal

メラノイジンに関する食品化学的研究

本間清一
日本栄養・食糧学会誌 : Nippon eiyo shokuryo gakkaishi = Journal of Japanese Society of Nutrition and Food Science 58(2), 85-98, 2005-04-10
食品の褐変により生じた色素 (褐色色素) であるメラノイジンの性質と主な前駆物質を食品ごとに明らかにした。褐変の原因として一般性が高いグルコースとアミノ酸から調製したモデルメラノイジンを材料に, 焦点電気泳動パターン, 金属キレート能, レクチン親和性, 酸化・還元, 調製時のpH条件, ラットにおける出納試験を調べた。Streptomyces werraensis TT14, Paecilomy …
NAID 10020369768

Isolation and Characterization of the K5-Type Yeast Killer Protein and Its Homology with an Exo-β-1, 3-glucanase

Izgu Fatih,Altinbay Demet
Bioscience, biotechnology, and biochemistry 68(3), 685-693, 2004-03-23
… The pI value of the K5-type killer protein was measured at pH 3.7 by high voltage vertical gel electrofocusing. …
NAID 130000030820

Synergistic Antifungal Activity of Chitinolytic Enzymes and Prodigiosin Produced by Biocontrol Bacterium, Serratia marcescens Strain B2 against Gray Mold Pathogen, Botrytis cinerea

SOMEYA Nobutaka,NAKAJIMA Masami,HIRAYAE Kazuyuki,HIBI Tadaaki,AKUTSU Katsumi
Journal of general plant pathology : JGPP 67(4), 312-317, 2001-11-25
… Two major enzymes, a 58-kDa endochitinase and a 98-kDa chitobiase, were purified by chromatography and electrofocusing, respectively. …
NAID 110002682988