Isoelectric focusing
Encyclopedia
Isoelectric focusing, also known as electrofocusing, is a technique for separating different molecules by their electric charge
differences. It is a type of zone electrophoresis
, usually performed on protein
s 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.
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 protein
s, which separate based on their relative content of acidic and basic
residues
, whose value is represented by the pI. Proteins are introduced into a 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. 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
.
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.
since it has the potential to provide rapid protein analysis, straightforward integration with other microfluidic unit operations, whole channel detection, smaller sample sizes and lower fabrication costs.
Electric charge
Electric charge is a physical property of matter that causes it to experience a force when near other electrically charged matter. Electric charge comes in two types, called positive and negative. Two positively charged substances, or objects, experience a mutual repulsive force, as do two...
differences. It is a type of zone electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
, usually performed on protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s in a gel
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
, that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
of its surroundings.
Isoelectric focusing in laboratory
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
Isoelectric point
The isoelectric point , sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge....
(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
Electric current
Electric current is a flow of electric charge through a medium.This charge is typically carried by moving electrons in a conductor such as wire...
is passed through the medium, creating a "positive" anode
Anode
An anode is an electrode through which electric current flows into a polarized electrical device. Mnemonic: ACID ....
and "negative" cathode
Cathode
A cathode is an electrode through which electric current flows out of a polarized electrical device. Mnemonic: CCD .Cathode polarity is not always negative...
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 protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s, which separate based on their relative content of acidic and basic
Base (chemistry)
For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...
residues
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
, whose value is represented by the pI. Proteins are introduced into a Immobilized pH gradient
Immobilized pH gradient
Immobilized pH gradient gels are the acrylamide gel matrix co-polymerized with the pH gradient, which result in completely stable gradients except the most alkaline pH values. The immobilized pH gradient is obtained by the continuous change in the ratio of Immobilines.An Immobiline is a weak...
gel composed of polyacrylamide
Polyacrylamide
Polyacrylamide is a polymer formed from acrylamide subunits. It can be synthesized as a simple linear-chain structure or cross-linked, typically using N,N-methylenebisacrylamide. Polyacrylamide is not toxic...
, starch
Starch
Starch or amylum is a carbohydrate consisting of a large number of glucose units joined together by glycosidic bonds. This polysaccharide is produced by all green plants as an energy store...
, 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
Isoelectric point
The isoelectric point , sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge....
value by as little as 0.01. Isoelectric focusing is the first step in two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins...
, in which proteins are first separated by their pI and then further separated by molecular weight through SDS-PAGE
SDS-PAGE
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility...
.
Isoelectric focusing by living cells
According to some opinions, 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., phosphorylationPhosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
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
Microchip based electrophoresis is a promising alternative to capillary electrophoresisCapillary electrophoresis
Capillary electrophoresis , also known as capillary zone electrophoresis , can be used to separate ionic species by their charge and frictional forces and hydrodynamic radius. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an...
since it has the potential to provide rapid protein analysis, straightforward integration with other microfluidic unit operations, whole channel detection, smaller sample sizes and lower fabrication costs.