What is the principle of IEF?

Isoelectric Focusing or IEF is a method of separating proteins according to their Isoelectric points in a pH gradient. Isoelectric point denoted as pI is defined as the pH at which protein carry no net charge, or pH at which protein become immobile in an electric field.

What is the procedure of isoelectric focusing?

Procedure. 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.

How does IEF separate proteins?

IEF separates amphoteric compounds, such as proteins, with increased resolution in a medium possessing a stable pH gradient. The protein becomes “focused” at a point on the gel as it migrates to a zone where the pH of the gel matches the protein’s pI.

What is the importance of isoelectric point?

The isoelectric point is significant in protein purification because it represents the pH where solubility is typically minimal. Here, the protein isoelectric point signifies where mobility in an electro-focusing system is zero—and, in turn, the point where the protein will collect.

What are ampholytes in IEF?

An ampholyte is simply a water-soluble molecule that can act both as an acid and a base depending on pH (just like an amino acid!). The easy movement of the ampholytes through the gel matrix helps the sample molecules to move along the pH gradient with them.

What is ampholytes chemistry?

An ampholyte is a molecule containing both acid and base functionality (see 5.16 Ionization Constants and Ionization Profiles).

What are the advantages of isoelectric focusing?

IEF’s greatest advantage is its high resolution, resulting in greater separation of solutes. IEF of serum proteins results in many more bands; these bands are sharper because each pH region is very narrow. Performing IEF is easier because the placement of sample application is not important.

What do you mean by isoelectric focusing of a material?

Definition of isoelectric focusing : an electrophoretic technique for separating proteins by causing them to migrate under the influence of an electric field through a medium (such as a gel) having a pH gradient to locations with pH values corresponding to their isoelectric points.

What is isoelectric pH how is it useful in the separation of proteins?

4.6. Separation of proteins at the isoelectric point is called isoelectric focusing. In isoelectric focusing a gradient of pH and an electric potential are applied across the gel, making one end more positive than the other. Separation occurs on the basis of the positive or negative groups present on the molecule.

What affects isoelectric point?

The isoelectric point (pI) is the pH at which a particular molecule carries no net electrical charge. The net charge on the molecule is affected by the pH of its surrounding environment and can become more positive or negative due to the gain or loss of protons, respectively.

What happens at isoelectric point?

The isoelectric point (pI) is the pH of a solution at which the net charge of a protein becomes zero. At solution pH that is above the pI, the surface of the protein is predominantly negatively charged, and therefore like-charged molecules will exhibit repulsive forces.

What are ampholytes give example?

Ampholytes are amphoteric molecules that contain both acidic and basic groups. For example, an amino acid H2N–RCH–CO2H has both a basic group NH2 and an acidic group COOH, and exists as several structures in chemical equilibrium: H2N–RCH–CO2H + H2O ⇌ H2N–RCH–COO− + H3O+ ⇌ H3N+–RCH–COOH + OH− ⇌ H3N+–RCH–COO− + H2O.

What are the advantages of the ionic focus technique?

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 ).

How does isoelectric focusing work?

Isoelectric focusing works because charged molecules will migrate towards regions of opposite charge (the electrodes) when an electric potential is applied to them. [1] Force these molecules to migrate through an immobilized pH gradient and they will also migrate towards regions of opposite charge.

How do you focus molecules?

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.