It is the first-dimension electrophoresis in the 2-D PAGE. Isoelectric focussing was described briefly in the first blog, 2-Dimensional PAGE . In this blog, I would discuss it in more details.

To start with, the electrophoretic mobility depends on the pH of the compound and the surrounding. Proteins are amphoteric molecules, i.e. they can act as both acid and base because they can carry either positive, negative, or zero net charge depending on the pH of their surroundings.

Now, the net charge of a protein is the sum of all the negative and positive charges of its amino acid side chains and amino and carboxyl-termini.

The isoelectric point (pI) is the specific pH (of medium) at which the net charge of the protein is zero. Proteins become positively charged at pH values below their pI and negatively charged at pH values above their pI (see the fig).

Isoelectric focussing IEF pI point protein
Characteristics of protein at different pH

If the pH of the electrophoretic medium is identical to the pI of a protein, the protein has a net charge of zero and does not migrate toward either electrode. Hence if a mixture of proteins is run in a gradient of pH, the individual proteins will cease to move whenever the pH (of medium) is identical to their pI, thus getting separated on the basis of their pI.

The Isoelectric Focussing (IEF) is performed in a medium of gradually changing pH i.e. a pH gradient. Traditionally the pH gradient was prepared using carrier ampholytes. Carrier ampholytes are small, soluble molecules having both positive and negative charged groups with different pI. Under the influence of electric field they arrange in range of isoelectric points establishing a pH gradient as the ampholytes with highest pI will move to the anode and vice versa. IEF is carried out in carrier ampholyte- generated pH gradient in polyacrylamide gel rods in tubes. The proteins migrate within carrier ampholyte gradient, and ceases to move when it reaches the pI.

The disadvantage of this method is tendency of ampholytes to drift towards cathode, low mechanical stability, batch-to-batch variability, and the probability of the soft gel to be stretched or broken

Angelika Gorg,Walter Weiss and Michael J. Dunn introduced immobilized pH gradient. The pH gradient was generated using buffering monomers called Immobiline in the polyacrylamide gels. Immobiline is a weak acid or base defined having particular pl. Immobilized pH gradients (IPG) are made by mixing acrylamide mixture with Immobiline having acidic buffering property and basic buffering property together. The immobilized pH gradient in gels with plastic backing. This is much improved and simplified the separation process.

Isoelectric focussing IEF by The Biotech Notes
Separation of proteins by IEF

Commercially, a wide range of length and pI are available; for e.g. a strip length from 7 to 24 cm and a pI ranged from 3-10, 4-6, 5-7, 8-9 are produced by many commercial companies. Another available type of IPG strips produced by Amersham is non-linear (NL) series. For instance, 3-10 NL,18 or 24 cm which enables focusing of the majority of neutral proteins with pI from 5-7 and with a smaller focusing length to the extreme acidic (3-5) and basic (8-10) proteins.  In general, the IPG strips does not exhibit cationic accumulation and produces of better focused protein with less smearing.

IPG strips

(Image source: Biophoretics)

Non-equilibrium pH gel electrophoresis (NEPHGE) is a technique developed for resolving basics proteins with extremely high PI (~ 7.0 to 11.0) that cannot be resolved by conventional IEF due to their extreme PI nature.  In this technique, the proteins move at different rate across the gel owing to the charge and the volt hours setting determine the speed pattern and reproducibility. The procedure is similar to that in IEF but, the polarity of 1DE is reversed by using an adapter that attaches the power supply.

NEPHGE procedure separates proteins with basic PIs but it is best to use a wide-range ampholyte mixture. A basic mixture of ampholytes will crowd the acidic proteins in a narrow region and potentially obscure some of the basic proteins. For the NEPHGE procedure, the positions of the anode and cathode buffers are reversed. This results in a better separation of the basic proteins. During NEPHGE, the IEF gels are not actually “focused” to their pIs. The separation is based on the migration rates of the differentially charged polypeptides as they move across the gel. Therefore, it is necessary to pay strict attention to accumulated volt hours during the run to assure reproducible patterns in subsequent separations. During the pre-run, a pH gradient is set up, and the focusing voltage is reached. The resistance in the gels is such that this voltage should be achieved in <2 h. If it takes longer, the samples may have high conductivity or the ampholyte quality may be inferior. The conditions for optimum polypeptide separation will most probably have to be empirically determined for the NEPHGE gels. This is because the proteins are not focused to their respective pIs as in standard IEF. Therefore, a series of test runs with different accumulated volt-hours should be compared to optimize for each sample.

After proteins are isoelectrically focussed, the IPG strip is carefully washed and then reduced and alkylated. After alkylation, the IPG strip is placed on the edge of the prepared gel for the next dimension (SDS PAGE).

Hope you like this piece of information. Please let me know what you thing and if there is anything else that I can write on for you.

Would love to hear it from you.

Till then be happy and be healthy.

Previous posts on 2D PAGE:

2-Dimensional PAGE

Sample Preparation In 2-D PAGE

IPG strip Rehydration

Next posts:

Second-dimension: SDS-PAGE

Protein Visualization and Staining (2-D PAGE)



2-D Electrophoresis using immobilized pH gradients: Principles and Methods, Amersham Biosciences, 80-6429-60, Edition AC

Two-Dimensional Polyacrylamide Gel Electrophoresis – A Practical Perspective. Sameh Magdeldin, Ying Zhang, Bo Xu, Yutaka Yoshida, Tadashi Yamamoto.