QAE-sephadex

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... 

Table II. Carbohydrate compositions (weight percentage) of individual oligomer peaks purified (QAE-Sephadex or HPLC ion-exchange separation, respectively) from mixtures of citrus pectin oligomers or B fruit extracts Compositions shown are for peaks whose biological activity is described in Figure 4. Uronic acid values are based on colorimetric assay. Proportions of neutral sugars were determined by GC and adjusted so that totals equal 100%. In fact, some oligomers (G7 peaks 8, 9 and 10. B extract peak 10) produced small (less than 1 % of the total integrated area), unknown peaks in the GC chromatograms.

Fig. 3. QAE-Sephadex gradient separation of the B fruit extract. An 18 mg (uronic acid equivalents) sample of extract was dissolved in 20 ml of 125 mM imidazole-HCl buffer (pH 7.0) and applied to the column. The column was then eluted with 50 ml 125 mM buffer followed by a 125 mM to 1.5 M buffer gradient (500ml), and, finally, 50 ml of 1.5 M buffer. Fractions of 5 ml were collected and assayed for uronic acids. Groups of fractions (26-41, 45-50, 53-75 and 84-100) were pooled, concentrated by ultrafiltration and analyzed by HPLC.

The cyanide sensor developed by the authors group is based on the formation of an addition product between cyanide ion and pyridoxal-5-phosphate, and its subsequent retention in the sensor (a fluorimetric flow-cell packed with QAE-Sephadex resin). The eluent is not injected, but merged with a stream of 0.05 M HCl after the reactor that is used both to acidify the complex and elute it after measurement. The calibration graph for the target analyte was linear from 50 ng/mL to 3.0 pg/mL, and the relative standard deviation and sample throughput were 1.4% (for 2 pg CN7mL) and... 

QAE-Sephadex Diethyl-(2-hydroxyl- propyl)-aminoethyl Dextran Strong... 

Bovine lung or mucous QAE Sephadex A50 Proteases of Bacillus subtilis Proteolytic enzymes from pig pancreas... 

Resuspend the ammonium sulphate pellet in 1 ml PBS pH 6.5 and apply to a 20 ml column of QAE Sephadex A-50 (Pharmacia) equilibrated with the same buffer, which is also used to elute the column. Elution of antibodies may be detected by ELISA or by polyacrylamide gel electrophoresis (Campbell, 1984). 

Diethyl-(2-hydroxyl-propyl)- aminoethyl Dextran QAE-Sephadex... 

H]AMP has been incubated in the presence of MgCh in Tris-HCl buffer, pH 7.4, and the [ HJadenosine formed isolated for radioassay by fractionation on an ion exchange column of QAE Sephadex [181,182]. 

The sequential purification of T. borchgrevinki serum on a DEAE-cellulose ion-exchange column and a QAE-Sephadex A-25 column is seen in Figs. 4 and 5. Slab-gel electrophoretic patterns of the mixture of active AFGP components 1-5 and of the three main active... 

Fig. 5. Elution profile obtained when fraction I from two DEAE-cellulose columns (Fig. 4) were combined and chromatographed on a QAE-Sephadex A-25 column (2.3 x 70 cm). Fraction I was eluted with the starting buffer, 2.5 mM Tris, pH 9.5. A linear gradient to 0.1 M Tris-HCl at pH 9.5 was started at tube 25. Each compartment contained 1000 ml of buffer 13.5 ml were collected in each tube. At tube 160, the elution buffer was changed to 0.15 M Tris-HCl at pH 9.5. From DeVries et al. (1970), reproduced with permission.

Fig. 6. Acrylamide gel electrophoresis of glycoproteins separated by QAE-Sephadex column chromatography shown in Fig. 5. Strips of Whatman No. 3 filter paper, 1 mm X 1 cm, were dipped into 5% solutions of the glycoproteins and inserted into slots at the origin. The gel was stained for carbohydrate with a-naphthol and concentrated HjS04. The unlabeled fraction shows glycoproteins 1-5, which were chromatographed on the QAE-Sephadex column. Fraction III from Sephadex contained glycoprotein 3, fraction V contained glycoprotein 4, and fraction VII contained glycoprotein 5. From DeVries et al. (1970), reproduced with permission.

Alternatively, QAE-Sephadex A-50 may be rapidly equilibrated with ethylene-diamine (2.88 g/l)-acetic acid (73 ml of 1 M/1) buffer, pH 7.0, in a fume hood. The rabbit or human serum is diluted with an equal volume of the same buffer, and applied to the column. IgG passes through while other proteins are adsorbed. Contaminants may be desorbed with a buffer consisting of 435 ml 600 mM acetic acid and 130 ml 600 mM sodium acetate per litre (pH 4.0). Volume change of the ion-exchanger is avoided since the ionic strength of the buffer is maintained at 0.1 (Tijssen and Kurstak, 1974). The yield is 70-85% for the various sera with at least 90% purity if overloading is avoided. Not more than half the column volume of diluted serum should be applied. 

Small-scale protein purification protocol is suitable when seminal fluid or prostate tissue is used as starting material. Perform all the steps for small-scale protein purification in +4 °C (or cold room). Same buffers than in mass-scale purification are used. Prepare your own columns using strong anion exchanger matrix such as QAE Sephadex A-25, and for gel filtration matrix use e.g. Sephacryl S-200 HR. L-(+)-tartrate afiSnity column is prepared in the same way than for mass-scale purification method. Use peristaltic pumps, gradient mixer and fraction collector in +4 °C. 

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