Ampholytes isoelectric focusing

Isoelectric focusing takes along (from ca 3 to 30 h) time to complete because sample compounds move more and more slowly as they approach the pH in the gel that corresponds to their isoelectric points. Because the gradient ampholytes and the samples stop where they have no mobiHty, the resistivity of the system increases dramatically toward the end of the experiment, and the current decreases dramatically. For this reason, isoelectric focusing is usually mn with constant voltage. Constant current appHcation can lead to overheating of the system. 

Palusinski, OA Allgyer, TT Mosher, RA Bier, M Saville, DA, Mathematical Modeling and Computer Simulation of Isoelectric Focusing with Electrochemically Defined Ampholytes, Biophysical Chemistry 13, 193, 1981. 

Figure 3. Preparative isoelectric focusing. The PNL eluted from gel filtration was subjected to isoelectric focusing using a column of 110 ml capacity (LKB) with ampholytes pH 7-11. After 48 h (9.6 W constant power), fractions of 3 ml were removed and assayed for PNL activity (- - ) and pH (- -).

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Rabilloud, T., Valette, C., and Lawrence, J. J. (1994). Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients. Electrophoresis 15, 1552-1558. 

Two variations of the basic technique are isoelectric focusing and immuno-electrophoresis. The former offers improved resolution and sharper bands in the separation of weak acids, weak bases and ampholytes through the use of pH and density gradients superimposed along the potential gradient. The latter employs specific antigen-antibody interactions (Chapter 10) to visualize the separated components of serum samples. 

Finally, isoelectric focusing has been a useful extension of basic gel electrophoresis in protein analysis. In this technique, a series of ampholytes is placed on the slab via electrophoresis. An ampholyte is a substance whose molecule contains both acidic and basic functional groups. Solutions of different ampholytes have different pH values. Different ampholyte molecules differ in size and therefore will have varying mobilities in the electrophoresis experiment. Thus, these molecules migrate into the slab, take... 

Capillary isoelectric focusing (CIEF) is suitable for the separation of amphoteric analyses in a pH gradient. A continuous pH gradient is built up in the column by using ampholytes under a potential field. Amphoteric analyses migrate to the point where their net charge equal to zero and they form stationary and sharply focused zones. 

FIGURE 12 Application of capillary isoelectric focusing (clEF) for the determination of apparent p/ values of rMAb samples. Capillary Bio-Rad Bio-CAP XL capillary (50 pm x 24 cm) ampholyte 80% clEF Bio-Lyte Ampholyte 3-10 (2% solution with 0.5% TEMED, 0.2% HPMC) anolyte 20 mM phosphoric acid catholyte 40 mM sodium hydroxide focusing l5kV (625V/cm) for 5 min mobilization 20 kV (833V/cm) for 25 min with zwitterions (cathodic mobilizer from Bio-Rad) capillary temperature 25°C. (Reprinted from reference 40, with permission.)... 

Tests of Purity, Isoelectric focusing (lEF) in a 0.5-mm thick horizontal slab gel was performed with LKB pH 7-9 ampholyte (Ampholine 1809-136) (5). Electrophoresis was run at 10°C for 6 h at a constant voltage of 1800 V. Protein was visualized using silver stain (9) or Sigma Coomassie Brilliant Blue G-250. 

The status of the isoelectric focusing process can be followed by the current reading. When steady state is reached where no sample migration occurs anymore, the current drops to zero. After focusing, the ampholytes and solutes are mobilized again in order to pass the detector. Mobilization can be accomplished by replacing one of the solutions in the reservoirs at the capillary end with a salt (e.g., sodium chloride), or the volume in the capillary is pushed out by applying pressure. 

Isoelectric focusing is a procedure used to determine the isoelectric point (pi) of a protein (Fig. 3-21). A pH gradient is established by allowing a mixture of low molecular weight organic acids and bases (ampholytes p. 81) to distribute themselves in an electric field generated across the gel. When a protein mix-... 

FIGURE 3-21 Isoelectric focusing. This technique separates proteins according to their isoelectric points. A stable pH gradient is established in the gel by the addition of appropriate ampholytes. A protein mixture is placed in a well on the gel. With an applied electric field, proteins enter the gel and migrate until each reaches a pH equivalent to its pi. Remember that when pH = pi, the net charge of a protein is zero. 

An example of isoelectric focusing is shown at the left below. A mixture of seven proteins (and some impurities) was applied to a polyacrylamide gel containing a mixture of polyprotic compounds called ampholytes. Each end of the gel was in contact with a conducting solution and several hundred volts were applied across the length of the gel. The ampholytes migrated until they... 

Wallevik, K. 1973. Isoelectric focusing of bovine serum albumin. Influence of binding of carrier ampholyte. Biochim. Biophys. Acta 322, 75-87. 

---