SDS-PAGE is the standard technique used for separation of proteins in the lab, but that doesn’t meant that other techniques don’t have their place–one such technique is isoelectric focusing (IEF).
IEF, also known simply as electrofocusing, is a technique for separating charged molecules, usually proteins or peptides, on the basis of their isoelectric point (pI), i.e., the pH at which the molecule has no charge. IEF works because in an electric field molecules in a pH gradient will migrate towards their pI.
In most cases, a commercially available immobilized pH gradient (IPG) strip is used. The IPG strip consists of an acrylamide gel that contains wide pores to prevent a sieving effect based on protein mass, with a pH gradient. Various gradients are available, with wider gradients, such as pH 3-10 that are used for whole proteome analysis, and narrower ranges, such as pH 5-8 that are used for more specialist applications.
The sample is usually combined with carrier ampholytes to assist in migration. Ampholytes are a mixture of charged molecules with a range of pIs that matches the pI range of the IPG strip. The migration of the ampholytes encourages the sample molecules to move along the pH gradient. Ampholyte mixtures of a variety of pI ranges are commercially available.
After separation across the pH gradient, the sample is further separated (in 2D-PAGE) or analyzed (in the case of fractionation for mass spec–more on this below).
Applications of IEF
IEF is “traditionally” used as first stage separation for 2D-PAGE, separating proteins by charge prior to second dimension separation by SDS-PAGE. This additional separation allows resolution of a couple of thousand proteins on a 2D-PAGE gel–enough for the entire proteome of an organelle or bacterium. It can also be used to examine post-translation modifications of proteins.
Another use for IEF is for fractionation of proteins or peptides prior to mass spec. Previously, it was difficult to recover molecules separated by IEF. However, with the Agilent OFFGEL system, the proteins or peptides remain in solution rather than being trapped in the gel as with standard IEF. Electrofocusing of proteins in this way provides a convenient alternative to SDS-PAGE for sample fractionation prior to mass spec.
Using IEF to separate peptides also provides an alternative to strong cation exchange (SCX) fractionation, which is one of the most popular techniques for the separation of peptides prior to mass spectrometry. IEF separation of peptides has been shown to result in more peptide identifications from whole proteome samples than with SCX for some samples1.
Get specialized for practical applications!
A more specialist version of IEF is capillary IEF (CIEF). This is more challenging but can be a useful way to accurately determine a protein’s pI. It can also be useful for a variety of practical applications, such as accurate analysis of erythropoietin, QC of monoclonal antibodies, and for analysis of hemoglobin charge variants as an indicator of the severity of diabetes.
How it works in practice
Setting up an IEF experiment is probably easier than understanding the theory behind what’s actually happening!
- The IPG strips are rehydrated (face down) in a denaturing buffer (>6 M urea) with detergent, usually including carrier ampholytes (it’s a case of trial and error, but for many samples, it works better to add the ampholytes with the sample).
- There are 2 ways to actually load the sample–you either include it in the rehydration buffer or you load it in a small plastic cup at the end of strip (again, some samples work better with one or other way of loading–give both a try and see what works best for you).
- The strips are then placed in the IEF apparatus, filter paper wicks are placed over the ends of the gel (these collect salts and proteins/peptides that are outside of the pI range of the IEF strip).
- Electrodes are placed on top—you’re ready to run!
Voltages used differ greatly for different systems, so it is advisable to do your homework in advance and look it up in the manual (or ask a lab mate)—but make sure you use the right voltage for the length strip you are using!
Often IEF has to be run overnight, so go get some sleep. Then, the following day, you can come back and move onto the next step of your experiment. Have you got any tips for mastering IEF? Let us know in the comments below!
1 Mostovenko, E., Hassan, C., Rattke, J., Deelder, A. M., van Veelen, P. A., Palmblad, M., 2013, Comparison of peptide and protein fractionation methods in proteomics, EuPA Open Proteomics, 1: 30-37.Image Credit: Bart Everson