ISOELECTRIC FOCUSING

INTRODUCTION:

 

There are vast varieties of proteins in the biological world and thus separation based on either charge or mass is not efficient. This is because any two proteins can have the similar charge, mass or charge to mass ratio. However, their isoelectric point may differ. Isoelectric focusing is used to separate proteins based on their isoelectric point (pI).

 

Isoelectric focusing is an electrophoretic method in which proteins are separated on the basis of their pIs. It makes use of the property of proteins that their net charges are determined by the pH of their local environments. Proteins carry positive, negative, or zero net electrical charge, depending on the pH of their surroundings.

The net charge of any particular protein is the (signed) sum of all of its positive and negative charges. These are determined by the ionizable acidic and basic side chains of the constituent amino acids and prosthetic groups of the protein. If the number of acidic groups in a protein exceeds the number of basic groups, the pI of that protein will be at a low pH value and the protein is classified as being acidic. When the basic groups outnumber the acidic groups in a protein, the pI will be high with the protein classified as basic. Proteins show considerable variation in isoelectric points, but pI values usually falling the range of pH 3-12, many having pIs between pH 4 and pH 7.

Proteins are positively charged in solutions at pH values below their pI and negatively charged above their isoelectric points. Thus, at pH values below the pI of a particular protein, it will migrate toward the cathode during electrophoresis. At pH values above its pI, a protein will move toward the anode. A protein at its isoelectric point will not move in an electric field.

When a protein is placed in a medium with a linear pH gradient and subjected to an electric field, it will initially move toward the electrode with the opposite charge. During migration through the pH gradient, the protein will either pick up or lose protons. As it does, its net charge and mobility will decrease and the protein will slow down. Eventually, the protein will arrive at the point in the pH gradient equalling its pI. There, being uncharged, it will stop migrating. If a protein at its pI should happen to diffuse to a region of lower pH, it will become protonated and be forced toward the cathode by the electric field. If, on the other hand, it diffuses into a pH higher than its pI ,the protein will become negatively charged and will be driven toward the anode. In this way, proteins condense, or focus, into sharp bands in the pH gradient at their individual, characteristic pI values.

Focusing is a steady-state mechanism with regard to pH. Proteins approach their respective pI values at differing rates but remain relatively fixed at those pH values for extended periods. This type of motion is in contrast to conventional electrophoresis in which proteins continue to move through the medium until the electric field is removed. Moreover, in IEF, proteins migrate to their steady-state positions from anywhere in the system. Thus, the sample application point is arbitrary. In fact, the sample can be initially distributed throughout the entire separation system.

 

-Principle of Isoelectric focusing

The isoelectric point refers to the pH value where the protein carries a net neutral charge and will not move further in the influence of electric field. Protein is positively charged in solutions at pH below its pI and will migrate towards the cathode. Protein is negatively charged in solution at pH above its pI and will migrate towards the anode. At isoelectric pH, they will be in the Zwitter ion form with no net charge so the further movement will cease.

 

In isoelectric focusing, the conditions are set in a way that proteins will be separated exclusively based on their isoelectric point. pH gradient is established in gel by addition of ampholytes which increases the pH from anode to cathode.

 

INSTRUMENTATION AND REAGENTS:

 

 

-Establishing pH gradients

Stable, linear pH gradients are the keys to successful IEF. Establishment of such gradients is accomplished in two ways with two different types of molecules, carrier ampholytes and acryl amido buffers.

·         Carrier ampholytes (amphoteric electrolytes) are mixtures of molecules containing multiple aliphatic amino and carboxylate groups. They are small (about 300-1000 Da in size) multi-charged organic buffer molecules with closely spaced pI values and high conductivity. Ampholytes are included directly in IEF gels. In electric fields, carrier ampholytes partition into smooth pH gradients that increase linearly from the anode to the cathode. The slope of a pH gradient is determined by the pH interval covered by the carrier ampholyte mixture and the distance between the electrodes. The use of carrier ampholytes is the most common and simplest means for forming pH gradients.

·         Acrylamido buffers are derivatives of acrylamide containing both reactive double bonds and buffering groups. Their general structure is CH₂ = CH-CO-NH-R, where R contains either a carboxyl [-COOH] or a tertiary amino group [e.g., -N(CH₃)₂]. They are covalently incorporated into polyacrylamide gels at the time of casting. The key acrylamido buffers have pK values at pH 1, 3.6, 4.6, 6.2, 7.0, 8.5, 9.3, 10.3, and >12.

 

They can be used to cast just about any conceivable pH gradients. In any given gradient, some of the acrylamido compounds act as buffers while others serve as titrants. Published formulations and methods are available for casting the most common gradients. Because the buffering compounds are fixed in place in the separation medium, the gel sare called “immobi1ized pH gradients”, or IPGs. IPGs offer the advantage of gradient stability over extended runs. They are, however, more cumbersome and expensive to cast than carrier ampholyte gels. IPGs are commercially available in sheet form in a few pH ranges. A greater variety of pH ranges are available in IPGs that have been cut into strips for the IEF first dimension of 2-D PAGE.

 

IEF is a high-resolution technique that can routinely resolve proteins differing in pI by less than 0.05 pH unit. Antibodies, antigens, and enzymes usually retain their activities during IEF. The proper choice of ampholyte or IPG range is very important to the success of a fractionation. Ideally, the pH range covered by an IEF gel should be centered on the pI of the proteins of interest. This ensures that the proteins of interest focus in the linear part of the gradient with many extraneous proteins excluded from the separation zone.

 

With carrier ampholytes, concentrations of about 2% (w/V) are best. Ampholyte concentrations below 1% (w/V) often result in unstable pH gradients. At concentrations above 3% (w/V) ampholytes are difficult to remove from gels and can interfere with protein staining. When casting IPGs, follow published recipes and use buffering powers of about 3 meq throughout the gradient.

 

-Gels for isoelectric focusing

As an analytical tool, IEF is carried out in large-pore polyacrylamide gels (5%T, 3%C) which serve mainly as anticonnective matrices. Polyacrylamide IEF gels are polymerized with an initiator system including riboflavin for photopolymerization. Photochemical initiation of polymerization with a combination of the three compounds riboflavin, ammonium persulfate, and TEMED, results in more complete polymerization of IEF gels than does chemical polymerization in gels containing low-pH ampholytes. Suitable initiator concentrations are 0.015% ammonium persulfate, 0.05% TEMED, and 5 ting riboflavin—5’-phosphate. Photochemical polymerization is allowed to continue for 2 hr, with the second hour under direct lighting from a nearby fluorescent lamp.

 

 

The most common configuration for analytical IEF is the horizontal polyacrylamide slab gel. Gels are cast with one exposed face on glass plates or specially treated plastic sheets. They are placed on cooling platforms and run with the exposed face upward. Electrolyte strips, saturated with 0.1-1 M phosphoric acid at the anode and 0.1-1M sodium hydroxide at the cathode, are placed directly on the exposed surface of the IEF gel. Electrodes of platinum wire maintain contact between the electrical power supply and the electrolyte strips. In another possible configuration, the gel and its backing plate are inverted and suspended between two carbon rod electrodes without the use ofelectrolyte strips. IPG strips for 2-D PAGE are often run with the gel facing down in dedicated IEF cells.

Ultrathin gels (<0.5 mm) allow the highest field strengths and, therefore, the highest resolution of the analytical methods. Electrofocusing can also be done in tubes, and this configuration once constituted the first dimension of 2-D PAGE. Because of difficulties in handling and reproducibility with tube gels, IPG strips have largely replaced them.

Good Visualization of individual bands generally requires a minimum of 0.5 µg each with dye staining or 50 ng each with silver staining. One of the simplest methods for applying samples to thin polyacrylamide gels is to place filter paper strips impregnated with sample directly on the gel surface. Up to 25 µl of sample solution can be conveniently applied after absorption into 1-cm squares of filter paper. A convenient size for applicator papers is 0.2 X 1 cm, holding 5 µl of sample solution. Alternatively, 1- to 2-µl samples can be placed directly on the surface of the gel. In most cases, IPG strips (which are provided in dehydrated form) are rehydrated in sample-containing solution prior to electrophoresis. Rehydration loading allows higher protein loads to be applied to gels than do other methods. It is particularly popular because of its simplicity.

There are no fixed rules regarding the positioning of the sample on the gel. In general, samples should not be applied to areas where they are expected to focus. To protect the proteins from exposure to extreme pH, the samples should not be applied closer than 1 cm from either electrode. Forming the pH gradient before sample application also limits the exposure of proteins to pH extremes.

Precast IEF mini gels (6 cm long by 8 cm wide and 1 mm thick) are available for carrying out carrier-ampholyte electro-focusing. A selection of IPG sheets is also available for horizontal IEF. Vertical IEF gels have the advantage that the electrophores is equipment for running them is available in most laboratories and they can hold relatively large sample volumes. Because vertical electrophoresis cells cannot tolerate very high voltages, this orientation is not capable of the ultrahigh resolution of horizontal cells. To protect the materials of the electrophoresis cells (mainly the gaskets) from caustic electrolytes alternative catholyte and anolyte solutions are substituted in vertical IEF runs. As catholyte, 20 mM arginine, 20 mM lysine is recommended in vertical slab systems (0.34 g arginine free base and 0.36 lysine free base in 100 m1 of water). The recommended anolyte is 70 mM H₃PO₄, but it can be substituted with 20 mM aspartic acid, 20 mM glutamic acid (0.26 g aspartic acid and 0.29 g glutamic acid in 100 m1 of water).

-Power conditions and resolution in isoelectric focusing

The pH gradient and the applied electric field determine the resolution of an IEF run. According to both theory and experiment, the difference in pI between two resolved adjacent protein IEF bands (ΔpI) is directly proportional to the square root of the pH gradient and inversely proportional to the square root of the voltage gradient (field strength) at the position of the bands:

ΔpI∝ [(pH gradient)/ (voltage gradient)]²

Thus, narrow pH ranges and high applied—voltages give high resolution (small ΔpI) in IEF.

In addition to the effect on resolution, high electric fields also result in shortened run times. However, high voltages in electrophoresis are accompanied by large amounts of generated heat. Thus, there are limitations on the magnitudes of the electric fields that can be applied and the ionic strengths of the solutions used in IEF. Because of their higher surface-to-volume ratio, thin gels are better able to dissipate heat than thick ones and are therefore capable of higher resolution (high voltage). Electric fields used in IEF are generally of the order of 100 V/cm. At focusing, currents drop to nearly zero since the current carriers have stopped moving by then.

Figure 1  Peak broadening associated with narrowing pH gradient in IEF.

-Protein solubilization for isoelectric focusing

A fundamental problem with IEF is that some proteins tend to precipitate at their pI values. Carrier ampholytes sometimes help overcome pI precipitation and they are usually included in the sample solutions for IPG strips. In addition, non-ionic detergents or urea are often included in IEF runs to minimize protein precipitation.

Urea is a common solubilizing agent in gel electrophoresis. It is particularly useful in IEF, especially for those proteins that tend to aggregate at their pIs. Urea disrupts hydrogen bonds and is used in situations in which hydrogen bonding can cause unwanted aggregation or formation of secondary structures that affect mobilities. Dissociation of hydrogen bonds requires high urea concentrations (7-8 M). If complete denaturation of proteins is sought, samples must be treated with a thiol-reducing agent to break disulfide bridges (protein solutions in urea should not be heated above 30°C to avoid carbamylation).

 

Figure 2   Principle of isoelectric focusing (IEF).

 

High concentrations of urea make gels behave as if they had reduced pore sizes.This is because of either Viscosity effects or reductions in the effective size of water channels (pores). Urea must be present in the gels during electrophoresis, but, unlike SDS, urea does not affect the intrinsic charge of the sample polypeptides. Urea solutions should be used soon after they are made or treated with a mixed-bed ion-exchange resin to avoid protein carbamylation by cyanate in old urea.

Some proteins, especially membrane proteins, require detergent solubilisation during isolation. Ionic detergents, such as SDS, are not compatible with IEF, although non-ionic detergents, such as octylglucoside, and zwiter ionic detergents, such as 2-[(3-cholamidopropyl) dimethylarnmonio]-1-propane-sulfonate (CHAPS) and its hydroxyl analog CHAPSO, can be used. NP-40 and Triton X-100 sometimes perform satisfactorily, but some preparations may contain charged contaminants.

Concentrations of CHAPS and CHAPSO or of octylglucoside of 1-2% in the gel are recommended. Some proteins may require as high as 4% detergent for solubility. Even in the presence of detergents, some samples may have stringent salt requirements. Salt should be present in a sample only if it is an absolute requirement. Carrier ampholytes contribute to the ionic strength of the solution and can help to counteract a lack of salts in a sample. Small samples (1 to 10 ul) in typical biochemical buffers are usually tolerated, but better results can be obtained with solutions in deionized water, 2% ampholytes, or 1% glycine. Suitable samples can be prepared by dialysis or gel filtration.

RESULT:

 

-APPLICATIONS:

1) Widely used for separation and identification of serum proteins.

2) Used in food and agricultural industries, forensic and human genetics laboratories.

3) Used in enzymology, immunology and membrane biochemistry.

4) 2D Gel electrophoresis is an application of IEF. Protein is first separated based on pI and then based on molecular weight using SDS-PAGE.

-ADVANTAGES:

1) Proteins that by as little as 0.001 pH units can be separated.

2) As spreading of bands is minimized due to application of the applied field and the pH gradient, high resolution can be achieved.

3) Isoelectric focusing (IEF) is a powerful analytical tool for the separation of proteins.

4) Performing IEF is easier because the placement of sample application is not important

 

-DISADVANTAGES:

1) Carrier ampholytes are generally used in high concentration, a high voltage (upto2000v) is necessary. As a result, the electrophoretic matrix must be cooled which sometimes makes it difficult.

2) Limited stability of solutions.

3) Lot-to-lot inconsistency.

4) Inadequate purity for application as a standard.