Free solution isoelectric focusing

Currently, there are five major modes of operation of CE capillary zone electrophoresis (CZE), also referred to as free solution or free flow capillary electrophoresis micellar electrokinetic chromatography (MEKC) capillary gel electrophoresis (CGE) capillary isoelectric focusing (CIEF) and capillary isotachophoresis (CITP). Of these, the most commonly utilized capillary techniques are CZE and MEKC (Rabel and Stobaugh 1993 Issaq 1999 Smyth and McClean 1998). 

Shang TQ, Ginter JM, Johnston MV, Larsen BS, McEwen CN. Carrier ampholyte-free solution isoelectric focusing as a prefractionation method for the proteomic analysis of complex protein mixtures. Electrophoresis 2003 24 2359-2368. 

The anaerobic hybrid mixture is mixed with an oxygenated solution of Hp in a stopped-flow instrument. The absorbance is monitored for 20 seconds, and the resulting observed rate constant is measured at 0.20 0.02 s. This results in an assembly free energy, AGasm. of —9.1 O.lkcal/mol when combined with the consensus on constant in Equation 6 (9). This value is in excellent agreement with the results of the equilibrium low-temperature isoelectric focusing experiment. 

The paper describes some applications to large scale protein fractionation using a recycling isoelectric focusing apparatus. Separation is achieved in free solution without the use of supporting media. Various alternatives for the formation of the pH gradient are discussed and results of a computer simulation are presented. 

Isoelectric focusing in polyacrylamide gel today appears to be nearly the most popular version of this technique [142-145]. The essential components for the gel preparation are identical with those used with conventional polyacrylamide gel electrophoresis (see pg. 428). The only difference is that carrier ampholytes (2% w/w of the total gel volume) are incorporated into the gel solution before polymerization. The protein sample (free of salts) can be mixed with the sample gel solution or it can be loaded in the conventional way in a sucrose solution. In the latter case it is covered with a protective layer of ampholyte. 

This technique, although described in the literature, has been given little attention. Isoelectric focusing without stabilizing media can be done in an apparatus similar to Tiselius free zone electrophoresis [152]. Separation itself occurs in a horizontal quartz tube that is rotated at 40 rpm to counteract convective forces [153]. The pH gradient is evolved without the addition of ampholytes, however this method leads to either too steep or too shallow gradients and is therefore not practically applicable. Another alternative method for free solution isoelectric focusing is the application of polyethylene coils however this procedure makes use of ampholines [154]. 

Herr, A.E., et al.. On-chip coupling of isoelectric focusing and free solution electrophoresis for multidimensional separations. Ana/. Chem., 2003, 75 1180-1187. 

The 77 K absorption spectrum (a) and the 4th derivative (b) of the purified PSII reaction center complex prepared by isoelectric focusing in digitonin solution are shown in Fig. 2. The spectrum is well-resolved than in Triton preparation (1). Absorption in the red maximum region, as well as the peaks around 600 nm, is clearly separated into two components 670 and 680 nm in the red, which might be attributed to the accessory chlorophyll and P-680 plus pheophytin acceptor, respectively. The 77 K emission spectrum of Triton preparation (1) exhibits shoulders on both sides of the main peak at about 681 nm, originating from free chlorophyll and the aggregates. However, the contribution of these components was markedly reduced in the spectrum of the complex prepared by the present procedure a sharp emission peak at about 683 nm was observed in this case. 

Preparative isoelectric focusing systems in free solutions like recycling isoelectric focusing in rotating tubes [8] and multicompartment electrolysers employing isoelectric membranes [9] have been developed for prefractionation and purification of proteins from highly heterogeneous mixtures. 

Out of all the techniques named above, the gel capillary electrophoresis and capillary isoelectric focusing have become most popular for separation of DNA and proteins. As biotechnology derived drugs become more prevalent, these techniques have become very important for the pharmaceutical industry. For the conventioneil pharmaceutical industry, the most frequently used techniques are free solution capillary electrophoresis and micellar electrokinetic capillary chromatography. 

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