Preparative solution isoelectric focusing

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. 

Electrophoresis plays a key role as an analytical or preparative technique in the characterization of natural organic matter because it gives information about the behavior of these molecular mixtures in controlled solution conditions, depending on both the size and the charge distribution frequency of the analytes in the complex mixture. Historically, the first electrophoretic separations were conducted with environmental colloids and over the years all the techniques based on zone, gel electrophoresis, or isoelectric focusing were used in their different setups to analyze natural organic matter and environmental particles to a minor extent. The goal of... 

Capillary isoelectric focusing separates analytes based on differences in their isoelectric points (pi) using the same principles as in preparative solution IEF. After a focusing step, that builds up a linear pH gradient in the capillary (controlled with zwitterionic internal markers), the analytes move as a function of their respective charge until they reach a position of zero charge (isoelectric point). The solution is then mobilized in CIEF to the detector hydrodynamically. 

The gel solution was prepared as described in Section III, and 1 mg of myoglobin (horse) was added to 8 ml of it. The isoelectric focusing tube was filled with 7 ml of this solution, giving a working gel length of 8 cm. 

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. 

The pellet was solubilized with Triton X-100 at 4 C for 1 hour in the dark. After centrifugation, the supernant was mixed with a 2% solution of Servalyt 3-6 (Serva, FRG) in 20% glycerol and brought to 0.2% final cone, of Triton X-100. Preparative isoelectric focusing was performed at 4 C ( Rotophor Cell) for 4 hours with 12 Watt. At pH 4.2-4.4 a green band was focused, which consisted of PSI complexes. 

Japanese Pharmacopoeia (JP), and the EP. In addition, a CZE method is currently used as an identification test for EPO concentrated solution in the EP. This represents the first example of the use of CE for the monitoring of a biopharmaceutical by an official method. A second example is in the final stage of implementation and involves the determination by CZE of charge variants in somatropin preparations. This test, which was found to provide more precise quantitative data, will replace the current isoelectric focusing method. 

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. 

GDH apo-enzyme and PQQ were obtained as described by Van der Meer(2). 500 of the apo-enzyme (Img in a phosphate buffer, pH 7.0) was reconstituted by addtion of 10 pi of the PQQ (5 mg ml in 10 mM HEPES with 3 mM CaCl2, pH 8.0), and the mixture was incubated at room temperature for 2 hours. The enzymatic activity of the reconstituted holo-enzyme was assayed spectrophotometrically by monitoring the decoloration of Wurster s Blue (J). The reconstituted GDH (in its reconstitution solution) can be stored at 4 C for more than two months without measurable loss of activity. The isoelectric focusing electrophoresis was done using the procedure of Katakis, Davidson and Heller (Katakis, I. Davidson, L. Heller, A. in preparation for publication). 

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