A Trusted Guide for Successful Isoelectric Focusing

Isoelectric Focusing

Isoelectric focusing techniques are widely used for the separation, characterizing and analyzing of peptides, proteins, cell membranes proteins, lipoproteins, glycol-proteins, enzymes, and isoenzymes. IEF can be powerful in investigating the factors that influence the net molecular charge of complex macromolecules or single molecules. 

This technique allows us to analyze the ligand binding to proteins and research on the subunit structures in the multimeric proteins.  Different applications will require the use of different isoelectric focusing techniques for ideal results. This is why we have a wide variety of commercially available products for isoelectric focusing. To ensure successful analysis using isoelectric focusing, you should consider the following.

Horizontal gels

IEF equipment is carried out in horizontal gels because of several reasons. First, the IEF separates according to their charge, and the gel matrix should have large pore sizes. Such a soft gel will slide down perfectly between the vertical glass plates. This gel must be cast or spread on a film support.

Samples

For excellent results, the samples should be applied on a well-defined pH location on the pH gradient so as to avoid isoelectric focusing in immobilized pH gradient of proteins. Precipitation and aggregation of some proteins is possible only when you use a horizontal gel that is on an open surface.

Temperature control

For consistent results, isoelectric focusing necessitates exact temperature control and efficient cooling. You can achieve excellent temperature control using horizontal ceramic cooling plates that are attached to a Peltier cooling plate or thermostatic circulator. For more on temperature control, check out mybiosource.com.

High Voltage

If you want sharply focused bands, then you should use high voltages in your isoelectric focusing experiments. This is because you can only get sharply focused bands when you use a highfield strength. When using high voltages, be sure to utilize horizontal equipment because they are the only ones that can satisfy the necessary safety precautions.

Power supply

You must ensure that your power supply provides adequate voltage for attaining sharply focused Isoelectric focusing zones and it should be programmable to automatically run a multiphase IEF protocol. Modular systems are currently available for running normal sized IEF techniques of about 10cm or longer separation distances. IEF in mini gels or 4cm separation distances can be carried out in an automated electrophoresis unit that contains a separation chamber, staining unit, and programmable power supply.

Use additional techniques


You can increase the power of resolution of the isoelectric focusing technique by putting together or running a two-dimensional procedure. You can use IEF with other methods like gel electrophoresis, immunoelectrophoresis and isotachophoresis. You can also couple the resolution of HPLC to IEF or use electrophoresis in the denaturing medium using denaturing agents such as sodium dodecyl sulfate or urea. The use of different biochemical techniques results in exceptional separation and purification of proteins, viruses, bacteria, cells, and animalcules.



Isoelectric focusing is a scientific research technique that will be used for many years to come. This is because it enables excellent separation and study of proteins and other molecules. The Isoelectric focusing method is also appropriate for preparative applications that necessitate purification of large amounts of proteins with over one hundred micrograms.
A Trusted Guide for Successful Isoelectric Focusing A Trusted Guide for Successful Isoelectric Focusing Reviewed by Ravish kumar on January 06, 2018 Rating: 5

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