Ampholytes separation from proteins

There are a number of ways to separate ampholytes from proteins. Electroelution ammonium sulfate precipitation and gel filtration, ion exchange, and... 

IEF was first successfully applied to proteins in 1938, when it was used to separate the protein hormones vasopressin and oxytocin from tissue extracts. Twenty years later, ampholytes were first focused in a continuous pH gradient, stabilized by a dense sucrose medium, as an alternative to the multicompartment method. The continuous pH gradient in the sucrose medium was established by allowing acid and base to diffuse into opposite ends of the sucrose medium, held in a U-cell, from their respective electrode chambers. The stabilization of this continuous pH gradient with carrier ampholyte species led to modern IEF methods. 

Vesterberg (50) studied the separation of carrier ampholytes from proteins by dialysis and by gel filtration. He used ampholytes containing... 

In the same work, Vesterberg showed that sucrose from the density gradient, as well as the carrier ampholytes, can be separated from the proteins simply and easily by gel filtration through Sephadex (AB Pharmacia, Uppsala, Sweden). Figure 16 shows a separation performed on Sephadex G50 with a column 20 cm high. The flow rate was 15 ml per... 

FIGURE 6 Separation of proteins by two-step CiEF using electrophoretic mobilization. The ampholytes generated a gradient from pH 3 to 10 after a focusing time of 300 sec in a neutral-coated capillary. Cathodic mobilization was initiated by replacing the catholyte (40-mM NaOH) with an alkaline zwitterion solution, pi, Isoelectric point. 

The proper choice of ampholyte range is very important to the success of an IEF fractionation. Ideally, the pH range covered by the focused carrier ampholytes should be centered on the pis of the proteins of interest. This insures that the proteins of interest focus in the linear part of the gradient with many extraneous proteins excluded from the separation zone. Carrier ampholyte concentrations of about 2% (w/v) are best. Concentrations of ampholytes 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. 

A representative IEF purification with the Rotofor that demonstrates the effectiveness of refractionation is shown in Fig. 8. The starting material for this fractionation was 150 mg of crude snake venom containing 2% (w/v) pH 3-10 carrier ampholytes. Aliquots of each of the 20 fractions from the run were analyzed in polyacrylamide IEF gels in pH 3-10 gradients. The focusing patterns of the proteins in the odd-numbered fractions are shown in Fig. 8a. The protein of interest (outlined with an oval) was found in fractions 10, 11, and 12. These three fractions were pooled, diluted with water to the volume of the separation chamber and refractionated. No additional ampholytes were added to the pooled material. IEF analysis of the refractionated material (Fig. 8 b) revealed that fraction 13 contained the protein of interest in nearly pure form. 

After labeling, 2xlysis buffer [8 M urea, 4% (w/v) CHAPS, 2% (v/v) carrier ampholytes, 2% (w/v) DTT] can be added to the samples in a l-tl dilution for lEF. Combining the three samples to be separated in one lEE strip or tube gel results in a total protein concentration of 150 pg per gel (50 pg Cy3-i-50pg Cy5-t50pg Cy2 labeled). From here methods are virtually identical to classical 2-DE. A peculiarity is that glass cassettes should have low intrinsic fluorescence capacity, since the gel will be scanned still assembled between the plates. 

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