Activation affinity chromatography

EC 3.4.24.3]. Purified by using /V-ethylmaleimide to activate the enzyme, and wheat germ agglutinin-agarose affinity chromatography [Callaway et al. Biochemistry 25 4757 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.]... 

Subtilisin (from Bacillus subtilis) [9014-01-1 ] [EC 3.4.21.62]. Purified by affinity chromatography using 4-(4-aminophenylazo)phenylarsonic acid complex to activated CH-Sepharose 4B. [Chandraskaren and Dhai Anal Biochem 150 141 7955]. 

Active derivatives of forskolin include 7-deacetyl-forskolin (EC5o 20(llM), 6-acetyl-7-deacetyl-forskolin (EC5o 40(llIVI), 7-deacetyl-7-0-hemisuccinyl-forskolin (EC50 50(llIVI). The last of these has been used as an immobilized affinity chromatography ligand for the purification of adenylyl cyclases from tissues. 

In a preliminary report, Ross et al. [40] used affinity chromatography to identify a putative bovine renal brush border Na /H exchanger. Brush border membranes were solubilized with Triton X-100 and chromatographed sequentially over lentil lectin Sepharose 4B and 5-(A-benzyl-iV-ethyl)amiloride coupled to epoxy-activated Sepharose 6B. The eluant contained 178- and 146-kDa proteins that were susceptible to Endo-F. Moreover, the eluants reacted on dot blot immunoassays with antisera to a 20-amino acid peptide of a human Na /H exchanger vide infra). The relationship between these proteins and the 66-kDa protein previously identified by the same investigators using amiloride photolabeling is presently unclear. 

This method was used, for example, for the solid-phase immunoassay of thyroxine (affinity chromatography). Various activation methods (CDI, periodate, and cyanogen bromide procedures) were compared with each other for coupling antibodies to magnetizable cellulose/iron oxide solid-phase particles. 211]... 

Bis-hydrazide-containing molecules also can be used to activate soluble polymeric sub-stances-containing aldehyde groups. For instance, dextran may be periodate oxidized to create numerous formyl functionalities on each molecule. Subsequent reaction with a homobifunctional hydrazide in large excess results in a hydrazide-activated polymer having multivalent-binding capability toward aldehydes or ketones (Chapter 25, Section 2.2). Insoluble support matrices suitable for affinity chromatography have been activated in a similar fashion to create the hydrazide derivative (O Shannessy and Wilchek, 1990). 

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