Table of Contents
What do you mean by isoelectric focusing?
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 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.
Does isoelectric focusing use SDS?
Classical sample preparation for IEF relies on non-ionic or zwitterionic reagents to disrupt protein complexes and denature proteins to ensure that the subsequent electrophoretic separations are carried out on polypeptide monomers. Since IEF separates proteins based on isoelectric point, SDS is not normally used.
What is the difference between isoelectric focusing and SDS-PAGE electrophoresis?
Isoelectric focusing (IEF) separates proteins on the basis of their isoelectric point. SDS-PAGE separates denatured proteins on the basis of mass only. SDS-PAGE can also be used as the second dimension of 2-D gel electrophoresis, and is also the starting point for Western blotting.
What is meant by isoelectric point what is its importance?
The isoelectric point (pI) is the pH value at which the molecule carries no electrical charge. The concept is particularly important for zwitterionic molecules such as amino acids, peptides, and proteins.
What is isoelectric focusing (IEF)?
Isoelectric focusing (IEF) is one of the most commonly used techniques for the separation of proteins. IEF separations are based on the pH dependence of the electrophoretic mobilities of the protein molecules. Isoelectric focusing makes use of electrical charge properties of molecules to focus them in defined zones in a separation medium.
What is isoelectric focusing in microbiology?
Isoelectric focusing (IEF) affords superior resolution of closely migrating proteins or various forms of a single protein that differ in charge owing to minor modifications. With this technique proteins migrate through a gel containing a pH gradient established with a mixture of carrier ampholytes (amphoteric oligoamino–oligocarboxylic acids).
What is isoelectric focused gel electrophoresis?
Isoelectric focusing is the first step in two-dimensional gel electrophoresis, in which proteins are first separated by their pI value and then further separated by molecular weight through SDS-PAGE.
What is iselectro focusing used for in immunology?
The technique is very useful when combined with immunoblotting; this enables the precise antigen-binding profile of an antiserum or monoclonal antibody to be established. Isoelectric focusing has been used to demonstrate the microheterogeneity of immunoglobulins.