Sepharose columns, elution

Sepharose column dialysis buffer 25 mM Na—MES, pH 6.0, 25 mM NaCl Sepharose column elution buffers 25 mM Na—MES, pH 6.0, with either... 

Purified by ammonium sulfate pptn, then fractionated on Sephadex G-75 column, applied to a Blue Sepharose column and eluted with ImM dihydrofolate. [Al Rubeai and Dole Biochem J 235 301 1986.]... 

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.]... 

Ricin (toxin from Castor bean Ricinus communis) [A chain 96638-28-7 B chain 96638-29-8] Mr -60,000, amorphous. Crude ricin, obtained by aqueous extraction and (NH4)2S04 pptn, was chromatographed on a galactosyl-Sepharose column with sequential elution of pure ricin. The second peak was due to ricin agglutinin. [Simmons and Russell Anal Biochem 146 206 1985.) Inhibitor of protein synthesis. EXTREMELY DANGEROUS, USE EXTREME CARE [instructions accompany product]. 

Purified by a [anti-human amniotic fluid-TIMP]-Sepharose immuno-affinity column eluted with 50mM glycine/HCl pH 3.0 buffer that is 0.5M in NaCl then by gel film [Cawston et al. Biochem J 238 677 1986]. 

Imidazole-HCl soluble polysaccharides Most of the polysaccharides of this group didn t elute from the Q-Sepharose column with 10 mM buffer (Table 3). The glycosyl composition (Table 4) shows that these polysaccharides are homogalacturonans and ramnogalacturonans with... 

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. 

The dissimilar multiprotein complexes, ALP-Fab and ALP-E3-Fab , were similarly purified over an octyl-Sepharose column. The protein fractions eluted from this column were characterized relative to the effective protein concentrations required to generate a specific response equivalent to that of ALP-Fab. These results show (Table 19.4) that the more hydrophobic protein fraction... 

The supernatant was collected and applied to a Q Sepharose column. The column was washed with buffer A and bound proteins were eluted with a gradient of buffer A to buffer B. Activity (measured as described in step 5) eluted at 100-150 him (NH4)2S04 and active fractions were pooled, yielding a pure protein as judged by SDS-polyacry-lamide gel electrophoresis. 

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. ... 

A method based on a metal chelate affinity chromatography (MCAC) was described by Carson (24). Generally, it consists of extraction/precipitation with succinate buffer followed by cleanup on a Chelating Sepharose column preloaded with copper(II) sulphate when TCs are specifically adsorbed by chelatation with metal ions bound. Elution of TCs was achieved using EDTA-containing buffer. 

NAD+ affinity tag. By offering an NAD+-laden column, or more often a 2-ADP-Sepharose column, to an NAD+-dependent enzyme such as a dehydrogenase, such enzymes are bound while all others are passing through the column. Subsequent elution with 0.5 mM NAD+ after a washing step to remove unspecifically bound protein results in a relatively pure fraction of protein which should contain only NAD+-dependent proteins. 

Figure 4. Elution patterns for rabbit antisera samples from a lactosyl sepharose column A = preimmune serum, B = anti-S -faecalis serum, C = anti-Gal-BSA serum and D — anti-Lac-BSA serum...

Anti-a-D-mannose antibodies have been isolated from the serum of rabbits immunized with the glycoconjugate of a-D-mannose and bovine serum albumin [85], The antibodies were purified by affinity chromatography with adsorption on a mannosyl-Sepharose column and elution with a-D-mannose or methyl a-D-mannoside. Such antibodies should be especially useful for studying the detection of diseases due to the appearance of abnormal glycoproteins that contain mannose polymers. 

The protein solution, previously dialyzed against 25 mM Tris/HCl, pH 8.0 containing 50 mM NaCl, or adjusted to pH 7.0 and NaCl added to 50 mM, is applied to the Heparin Sepharose column. The coulmn is washed with two volumes of loading buffer, and the adsorbed protein eluted with a linear gradient of 0.05-1 M NaCl. The pooled fractions are dialyzed and lyophilized as described above. 

A mixture of lentil lectin-reactive glycoproteins from pig lymphocyte-plasma membrane was isolated by lentil lectin-Sepharose chromatography of sodium deoxycholate-solubilized membranes.445 Eighty-seven percent of the protein applied (17% of hexose) passed through unretarded, and 13% of the applied protein (83% of hexose) was bound, and eluted with methyl a-D-glucopyranoside solution. Recovery was 95% of the material applied, in contrast to the recovery in similar experiments conducted on con A-Sepharose columns (80% recovery).850 The eluate from the lentil column, which contained at least ten glycoproteins, blocked lymphocyte transformation induced by lentil or kidney-bean lectins.445... 

Figure 7-14. Elution of lactate dehydrogenase (LDH) isozymes with a concave gradient of NADH. Protein, 0.2 mg, in 0.2 ml O.IM sodium phosphate buffer, pH 7.0, ImM /3-mercaptoethanol, and IM NaCl was applied to an AMP analogue-sepharose column (140 X 6 mm, containing 2.5 g wet gel) equilibrated with O.lM sodium phosphate buffer, pH 7.5. The column was washed with 10 ml of the latter buffer, then the isozymes were eluted with a concave gradient of 0.0 to 0.5mM NADH in the same buffer, containing ImM /3-mercaptoethanoI. Fractions of 1 ml were collected at a rate of 3.4ml/hour. The hatched area indicates the pooled fractions that were rechromatographed. [From P. Brodelius and K. Mosbach, FEBS Lett, 35 223 (1973).]...

---