Column chromatography DEAE-Sephadex

Step 4. Anion-exchange chromatography on a column of DEAE-Sephadex A-50. The column had been equilibrated with the basic buffer containing 0.12 M NaCl. The NaCl in the photoprotein solution was removed by gel filtration, and then the solution was added onto the column. The photoprotein adsorbed on the column was eluted with the equilibration buffer. 

Figure 3. Chromatography of native and ozonized lysozyme on DEAE Sephadex. Samples of lysozyme at the three different pHs were inactivated 95-100% by ozone. The samples were applied to a column of DEAE-Sephadex A-50 (0.8 X 56 cm) and eluted with O.OIM Tris-HCl pH 8.3 and a gradient of O.OIM NaCl. Fractions of 2 ml were collected. The results of four columns are plotted.

Fig. 5.1. Fractionation of heptanucleotides. Heptanucleotides were isolated from a pancreatic ribonuclease digest of l2 phage RNA by chromatography on DEAE-ceilulose. Fig. 4.1. They were dialysed against distilled water, loaded onto a 9 x 500 mm column of DEAE-Sephadex previously equilibrated with 7 M urea+ I mM EDTA + HCl to pH 2.7 and eluted with a linear gradient from 0 to 0.2 M NaCI in 7 M urea+1 mM EDTA + HCI to pH 2.7. The eluent was collected directly into polyethylene vials and the Cerenkov radiation from the label in each fraction was measured in the liquid scintillation counter.

Mitsuhashi and coworkers found streptomycin phosphate transferase in Pseudomonas aeruginosa TI-13 they isolated the inactivated compound, which was shown to have the empirical formula of a mono-0-phosphonodihydrostreptomycin monocarbonate trihydrate by elementary analysis. They also found the same enzyme in other strains, namely, 99, 137, 138, 351, 10126, Cape 18, and TI-11. The enzyme in the supernatant liquor (600 ml) from centrifugation of disrupted eells of strain TI-13 at 105,000g was precipitated by ammonium sulfate at 33-66% saturation. Further purification by successive, column chromatography on Sephadex G-75, DEAE-Sephadex A-50 (with a gradient of potassium... 

A proteinaceous inhibitor of rubber biosynthesis was purified from the C-serum of Hevea brasiliensis latex. The protein inhibited the incorporation of isopentenyl diphosphate into rubber. Purification was achieved by employing three column chromatography methods Sephadex G-150 gel filtration, DEAE-Cellulose ion exchange chromatography and Phenyl Sepharose CL-4B hydrophobic interaction chromatography. 21 refs. 

Schott additionally demonstrated that one gram PVA can be loaded with up to 10 mmol of mononucleotide [34]. For this example he u as solvent mixtures of pyridine and hexamethylphosphorotriandde and noted condensation yields of more than 90%. Purification and removal of excess reagents was done by dialysis, precipitation or separation by column chromatography on Sephadex or DEAE-cellulose. 

Purified by Sephadex G-200 filtration and DEAE-cellulose column chromatography. Hexosaminidase A was further purified by DEAE-cellulose column chromatography, followed by an ECTEOLA-cellulose column, Sephadex-200 filtration, electrofocusing and Sephadex G-200 filtration. Hexosaminidase B was purified by a CM-cellulose column, electrofocusing and Sephadex G-200 filtration. [Srivastava et al. 7 Biol Chem 249 2034 1974.]... 

Coiicin E (from E.coli) [11032-88-5], Purified by salt extraction of extracellular-bound colicin followed by salt fractionation and ion-exchange chromatography on a DEAE-Sephadex column, and then by CM-Sephadex column chromatography [Schwartz and Helinski J Biol Chem 246 6318 1971],... 

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

Thrombin (from bovine blood plasma) [9002-04-4] Mj 32,600 [EC 3.4.4.13]. Purified by chromatography on a DEAE-cellulose column, while eluting with O.IM NaCl, pH 7.0, followed by chromatography on Sephadex G-200. Final preparation was free from plasminogen and plasmin. [Yin and Wessler J Biol Chem 243 112 796S.]... 

Dried shrimp was ground, defatted with benzene, and then extracted with cold water. The luciferase extracted was purified first by a batch adsorption onto DEAE cellulose (elution with 0.4 M NaCl), followed by gel filtration on a column of Sephadex G-150, anion-exchange chromatography on a column of DEAE-cellulose (gradient elution 0.05-0.5 M NaCl), and gel filtration on a column of Ultrogel AcA 34. The specific activity of the purified luciferase was 1.7 x 1015 photons s 1 mg-1, and the yield in terms of luciferase activity was about 28%. 

Att eZcven y-cJuiin voA nts, discovered thus far, exhibit a change In electrophoretic mobility, and starch gel electrophoresis Is the recommended method for their detection. Quantitation of the variant can best be done by chromatography on columns of either DEAE-Sephadex or CM-Cellulose. The quantities of some variants In heterozygotes differ greatly. For Instance, the relative amount (expressed In %F /Fxotal) varies from 20-25% (F-Malta-I) to 10-15% (most Y C >aln variants) to 5-6%... 

Chromatography of hemoglobins on columns of DEAE-Cellulose (DE-52 mlcrogranular, preswollen Whatman) often results In excellent separations of many variants, because the hemoglobins are eluted as sharp, narrow zones generally widely separated from each other The hemoglobin zones are eluted from the DE-52 columns at a distinctly higher pH value than from a similar DEAE-Sephadex column ... 

This electrophoretlcally fast-moving variant was readily Isolated by DEAE-Sephadex chromatography. Hybridization analyses with canine Hb confirmed the suggestion that the abnormality was located In the a-chaln. The a-chaln was separated from the 3-chaln on 1.7 X 15 cm columns of CM-Cellulose using the method of Clegg tt aJL ( ). The CM-Cellulose used was CM-52 (Reeve Angel, Clifton, N.J.) and the developers were 8 M urea-3-mercaptoethanol phosphate buffers, pH 6.7 - 7.1. 

In the purification of pectinesterase from the fruits of Citrus nat-sudaidai,61 fractional salting-out with ammonium sulfate was followed by chromatography on a column of DEAE-cellulose and by separation of the active fraction on Sephadex G-100. A preparation (purified solution) having a specific activity 460-fold greater than that of the original extract was obtained. Its homogeneity was checked by disc electrophoresis, and its amino acid content was determined and fundamental, kinetic data were obtained. 

Miller and Macmillan49 purified pectinesterase produced by Fusarium oxysporum f. sp. vasinfectum by chromatography on DEAE-Sephadex A-25, Sephadex G-75, CM-Sephadex C-50, and CM-cellulose. The homogeneity of the pectinesterase obtained was confirmed by disc electrophoresis an apparent molecular weight of 35,000 was estimated by its behavior on a column of Sephadex G-75 (Superfine). 

The pattern of metabolites in bile (animals only) and in urine have been investigated using column chromatography (Amberlite XAD 2 and Sephadex DEAE), tic and reversed phase HPLC in combination with radioactivity monitoring. 

Column Chromatography of Crude Toxin. The WSAP obtained from culture 350F was retained in the crude state for assay. The 266.2 mg of WSAP obtained from 350G was treated on successive columns of silicic acid, DEAE cellulose, and Sephadex G-15 and yielded a single semipurified toxic product (GT-4) of 23.1 mg or 9% of the starting crude extract (Table I). 

Chromatographic Analysis. The samples of native and ozonized lysozyme (lysozyme treated with ozone just to the point of complete inactivation) were analyzed by column chromatography. The column (0.8 X 56 cm.) containing DEAE-Sephadex A-50 (Cl form) resin, was equilibrated with 0.1 M Tris Cl buffer, pH 8.3, and loaded with about 2-U mg of protein. Aliquots eluted with 0.1 M Tris-Cl pH 8.3 were collected and absorbance at 278 nm was measured. The native lysoz3mie eluted earlier than the ozonized products. This difference may be assoicated with both aggregation of protein and ionic behavior of the residues. 

Separation and purification chromatography has to be applied before the quantitative chromatography. Some purification techniques applied are lEC, such as DEAE-Sephadex A-25 [571], weak anion-exchange column [572,573], strong anion-exchange column (564), cation-exchange column... 

Nucleotide thiophosphate analogues. The preparation and purification of [ HJATPyS, pHJGTPyS, s ITPyS (6-thioinosine), cl ITPyS (6-chloroinosine) and [ HJATPyS are described and the general purification was achieved by chromatography of the nucleotide thiophosphates in the minimum volume of H2O placed onto a DEAE-Sephadex A25 column and eluting with a linear gradient of triethylammonium bicarbonate (0.1 to 0.6M for G and I nucleotides and 0.2 to 0.5M for A nucleotides). [Biochim Biophys Acta 276 155 7972]. 

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