DEAE-Sepharose

Ceruloplasmin (from human blood plasma) [9031-37-2] Mr 134,000. This principle Cu transporter (90-90% of circulating Cu) is purified by precipitation with polyethylene glycol 4000, balchwise adsorption and elution from QAE-Sephadex, and gradient elution from DEAE-Sepharose CL-6B. Ceruloplasmin... 

EC 1.15.1.1]. Purified by DEAE-Sepharose and copper chelate affinity chromatography. The preparation was homogeneous by SDS-PAGE, analytical gel filtration chromatography and by isoelectric focusing [Weselake et al. Anal Biochem 155 193 1986 Fridovich J Biol Chem 244 6049 7969]. 

Figure 4-4. Use of SDS-PAGE to observe successive purification of a recombinant protein. The gel was stained with Coomassie blue. Shown are protein standards (lane S) of the indicated mass, crude cell extract (E), high-speed supernatant liquid (H), and the DEAE-Sepharose fraction (D). The recombinant protein has a mass of about 45 kDa.

Because plants present chlorophylls and carotenoids simultaneously, it may be useful to separate both groups from each other in a laboratory or preparative scale in order to avoid contamination in further purification steps, mainly when they are prepared in large amounts. Clean-up procedures using an open column packed with absorbents such as alumina, magnesia, polyethylene powder, powdered sucrose, DEAE-Sepharose, starch, cellulose, or MgO HyfloSupercel are good approaches. MgO HyfloSupercel in a proportion of 1 1 or 1 2 is the usual adsorbent. Sucrose and cellulose are interesting as they do not alter the chlorophylls, but they are tedious to work with. 

Figure 3. Anion-exchange chromatography on DEAE Sepharose of fraction I of the Sephacryl S300 fractionation of a) saponified MHR population A after degradation with RGase and after b) degradation of the non-saponified MHR population A with RGase and RGAEase ---------, uronic acid —, neutral sugars thin line, NaOAc gradient [39],...

Table 4. Sugar composition (mol%) of the xylogalacturonan fractions obtained after DEAE Sepharose anion-exchange chromatography of the Sephacryl S300 fractions I originating from RGase degraded saponified apple MHR population A (1-a and 1-Z>) and from RGase/RGAEase treated non-saponified apple MHR population A (2-a, 2-b, and 2-c).

Figure 2 Ion-exchange chromatography on DEAE-Sepharose CL-6B (elution by acetate buffer pH 4.8) of (a) dialysed water-soluble pectins from extruded citrus fibres (SME = 250 kWh/t) and (b) dialysed acid extracted pectins from the same raw material, (empty symbols neutral sugars full symbols= galacturonic acids)...

Figure 1. Elution profile of ChSS on DEAE Sepharose Fast Flow. Eluent molarity (. galacturonic acid ( ) and neutral sugars ( ).

Crude polysaccharide fraction (GL-2) was prepared from the leaves of P. ginseng by hot water extraction, ethanol precipitation and dialysis, and GL-2 was fractionated by Cetavlon precipitation and weakly acidic polysaccharide fraction (GL-4) was obtained[3]. GL-4IIb2 was purified from GL-4 by DEAE-Sepharose CL-6B as described previousely [3]. In order to remove the color-materials, GL-4IIb2 was further purified by Q-Sepharose (C1 form), and the major fraction, eluted with 0.3 M NaCl, was repurifled by gel filtration on Bio-gel P-30 column to obtain purified active polysaccharide, GL-4IIb2. ... 

In order to remove the residual PME the AE fraction was further fractionated on Mono S cation exchange column. Unlike the DEAE-Sepharose column, where PME elutes after the AE activity, the order of elution was reversed on the Mono S column (Fig. 1). The last step in the purification was a gel filtration (Sephacryl S-200) column. 

A flow injection optical fibre biosensor for choline was also developed55. Choline oxidase (ChOX) was immobilized by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) after adsorption on weak anion-exchanger beads (DEAE-Sepharose). In this way, the sensing layer was directly created at the surface of the working glassy carbon electrode. The optimization of the reaction conditions and of the physicochemical parameters influencing the FIA biosensor response allows the measurement of choline concentration with a detection limit of 10 pmol. The DEAE-based system also exhibited a good operational stability since 160 repeated measurements of 3 nmol of choline could be performed with a variation coefficient of 4.5%. 

To elucidate the intramolecular interaction within the hybrid, CaM-PDE hybrid with EDC was immobilized onto DEAE-Sapharose, because immobilized hybrids were expected to have no interaction with each other (inter-hybrid interaction). The CaM-PDE hybrid was immobilized to DEAE-Sepharose suspended in 100 mM glycylglycine buffer (pH 7.5). In the presence of Ca2+, the CaM-PDE hybrid showed the activity change in its bound form. The activity of the CaM-PDE hybrid in the absence of Ca2+ was ca.40% as compared to that in the presence of Ca2+. The normalized modulation in the immobilized form is comparable to that in the free form, although the total modulated activity was smaller in the DEAE-Sepharose immobilized form as presented in Fig.30. These results indicate that the selfmodulation of CaM-PDE activity can be performed in its immobilized form, as this modulation was caused by the CaM moiety in the hybrid (intrainteraction). If CaM and PDE are independently present in solution, CaM has to randomly access to PDE to modulate the enzyme. However, in the case of CaM-PDE distinct CaM molecules modulates the corresponding PDE molecules in intra-hybrid interaction. In other words, CaM concentration... 

Upon purification of the XDH from C. purinolyticum, a separate Se-labeled peak appeared eluting from a DEAE sepharose column. This second peak also appeared to contain a flavin based on UV-visible spectrum. This peak did not use xanthine as a substrate for the reduction of artificial electron acceptors (2,6 dichlor-oindophenol, DCIP), and based on this altered specificity this fraction was further studied. Subsequent purification and analysis showed the enzyme complex consisted of four subunits, and contained molybdenum, iron selenium, and FAD. The most unique property of this enzyme lies in its substrate specificity. Purine, hypoxanthine (6-OH purine), and 2-OH purine were all found to serve as reductants in the presence of DCIP, yet xanthine was not a substrate at any concentration tested. The enzyme was named purine hydroxylase to differentiate it from similar enzymes that use xanthine as a substrate. To date, this is the only enzyme in the molybdenum hydroxylase family (including aldehyde oxidoreductases) that does not hydroxylate the 8-position of the purine ring. This unique substrate specificity, coupled with the studies of Andreesen on purine fermentation pathways, suggests that xanthine is the key intermediate that is broken down in a selenium-dependent purine fermentation pathway. ... 

Figure 2. Anion exchange chromatogram of ionically bound phloem peroxidases on DEAE-Sepharose. Collected fractions were analyzed for their oxidase activity towards TMB (1), syringaldazine (2) and isopropylamine salt from / -fluoroferulic acid (3). Bo cationic peroxidases Bj and B2 anionic peroxidases. Column was equilibrated with 0.01 M phosphate buffer (pH 7.1). Fractions were eluted with a NaCl gradient (0-0.5 M) in the same buffer (0.01 M phosphate, pH 7.1).

FIGURE 11.7 Selective displacement of AVP on DEAE Sepharose Fast Elow. Column dimensions were 10 X 290mm load was 6.5 mL AVP feedstock/mL resin. The flow rate was 1 mL/min. (Reprinted with permission from Elsevier from Barnthouse, K.A. et ah, J. BiotechnoL, 66, 125, 1998. Copyright.)... 

Stipe powder of C. comatus (100 g) was extracted three times with 1 L 95% ethanol under reflux for 2 h to remove lipid, and the residue was extracted three times with 2 L distilled water for 2 h at 80 °C with intermediate centrifugation (2000 x g, 15 min). After concentrating the collected aqueous supernatants to 400 mL (reduced pressure at 40 °C), a precipitation was performed with 3 volumes of 95% ethanol. The precipitate was washed with ethanol and acetone, and then dried at 40 C, yielding crude polysaccharide material. Crude polysaccharide material was dissolved in 100 mL 0.2 M sodium phosphate buffer (pH 6.0), and after centrifugation the solution was applied to a DEAE-Sepharose CL-6B column. 

DEAE-Sepharose fast flow anion-exchange chromatography column (10 x 300 mm)... 

EC 1.15.1.1]. Purified by DEAE-Sepharose and copper chelate affinity chromatography. The preparation... 

Fractionated by passing through a DEAE-sepharose column in 10mM phosphate buffer, pH 5.8... 

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