Sephadex layers

E. Sephadex Sephadex layers are prepared from modified dextran gels for the separation of hydrophilic solutes such as nucleic acids and peptides. The mechanism of separation is partition chromatography governed by size exclusion in the swollen gel containing pores of controlled dimensions. The gels, a layer spreader and special equipment for developing layers are available from Pharmacia Fine Chemicals. 

Clean sample application is as important with CF as with SEC (see Section 5.2.2.1). In particular, the gel surface must be even. Giri (1990) recommends applying a layer (1 to 2 cm) of Sephadex G-25 coarse onto the PBE gel bed. The Sephadex layer should facilitate an even sample application and serve as a mixing chamber. It also prevents the surface of the ion exchanger gel from being disturbed. It is common practice to equilibrate the sample before... 

WiELAND and Determann [191] have separated lactate-dehydrogenase isoenzyme and AMP, ADP and ATP from one another on DEAE-Sephadex layers, using gradient elution chromatography (p. 90). 

Fignre 12. Preparative lEF in granulated Sephadex layers A) Small-scale separation B) Large-scale separation a) Electrode b) Filter paper pad soaked in electrode solution c) Cooling block d) Glas.s plate e) Gel layer f) Focused proteins g) Trough 1178). 

Radioiodide has also been separated from iodine-labeled proteins (human serum albumin and Bovine IgG) by thin-layer gel filtration on Sephadex G-200 and G-75 superfine layers (163). (Sephadex is a modified dextran.) The protein solutions were applied in amounts of 5-10 vJL to the Sephadex layers and the angle of slope of the layers was 15°. Elution was carried out with 0.2 M Tris buffer (Tris = tris (hydroxymethyl) aminomethane) at pH 8.0. The buffer contained Tween 80. Separations took 45-60 min on Sephadex G-75 and 3-4 h on Sephadex G-200 and were particularly good on the latter. 

Renilla luciferin and luciferyl sulfate. Luciferyl sulfate is extracted from the organisms with methanol. The extract is concentrated, defatted with benzene, and the aqueous layer is extracted with ethyl acetate. Luciferyl sulfate in the extract is purified by chromatography on a column of Sephadex LH-20 (Hori et al., 1972 Inoue et al., 1977a). Free luciferin is obtained by heating the sulfate in 0.1 N HC1 at 90° C for 50 sec, followed by extraction with ethyl acetate. The luciferin of Renilla thus obtained was found to be identical with coelenterazine (Inoue et al., 1977a Hori et al., 1977). 

To obtain reliable chromatograms in the final step of the determination of the analytes by LC or GC, it is important to remove interfering signals resulting from coelution of other compounds. To this end, a variety of techniques are applied for cleanup of the sample extract. The most effective procedures for sample cleanup for PAH measurements are partitioning between M, N-dimethylformamide/water/cyclo-hexane and LC on silica and on Sephadex LH 20. Other cleanup procedures include LC on alumina or XAD-2 and preparative thin-layer chromatography. 

Delincee and Radola100 used a commercial preparation, as well as fresh tomatoes, for the preparation, purification, and characterization of tomato pectinesterase. The tomatoes were pressed and then homogenized directly with ammonium sulfate at 70% saturation. The precipitate obtained was extracted with 0.3 M phosphate and repeatedly salted out with ammonium sulfate, and the product was separated on a column of Sephadex G-75. The pattern of separation was similar to that in preceding work.50,97 A detailed study of the size properties of pectinesterase was conducted by gel-filtration and sedimentation analysis.100 By column and thin-layer gel-filtration on Sephadex G-75, the approximate molecular weight of a number of preparations of tomato pectinesterase was determined, values of 24,000 and 27,000 being obtained. A possible interaction of the... 

In our laboratory crude preparations of aphantoxins and anatoxin-a(s) are extracted similarly except at the final stages of purification (Fig. 2). A Bio-gel P-2 column (2.2 x 80 cm) is used for aphantoxins gel filtration and a Sephadex G-15 (2.6 x 42 cm) column for ana-toxin-(s). Both toxins are eluted with 0.1 M acetic acid at 1.5 ml/ min. Fractions of aphantoxins from Bio-gel P-2 run are spotted on thin-layer chromatography plates (Silica gel-60, EM reagents) and developed according to Buckley et al. (1976) (31). The Rf values for the aphantoxins, saxitoxin and neosaxitoxin standards (Table 1) indicates that two of the aphantoxins (i.e. I and II) are similar to saxitoxin and neosaxitoxin. 

Method B. In a Schlenk tube 100 mg (0.11 mmol) of chlorotris(triphenyl-phosphine)rhodium(I), ClRh[P(C6H5)3]3, is dissolved in a mixture of 20 mL of toluene and 10 mL of tetrahydrofuran. Then 20 mL of an aqueous solution of 1.87 g (3.3 mmol) of tppts is added to this solution to form a lower layer. After 12 h of vigorous stirring, the reaction mixture is transferred to a separation funnel. The aqueous phase is separated and washed twice, in each case with 5 mL of methylene chloride. The organic phases are discarded. The pure compound is obtained by column chromatography of the water phase on Sephadex G-15. Yield 180 mg (82%). 

The purification of glutamine cyclotransferase from papaya latex has been carried out by Messer and Ottesen (116). A batch procedure was used for the removal of impurities by passage of a papaya latex extract through a thin layer of carboxymethyl-Sephadex the active protein was separated by selective elution. Additional purification was achieved by chromatography on a column of carboxymethyl-Sephadex and by gel filtration on Sephadex G-100. The purified enzyme was homogeneous by the criteria of paper electrophoresis, ultracentrifugation, and gel filtration on Sephadex G-100 columns and chromatography on carboxy-methyl- or DEAE-Sephadex. The electrophoretic behavior of the enzyme indicates that it is a basic protein with an isoelectric point near... 

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