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By M. Dean Savage (auth.), Dr. Thomas Meier, Dr. Falk Fahrenholz (eds.)

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Extra resources for A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis

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To avoid these difficulties, spacers between biotin and the protein-binding part of the ligand are introduced, which can be cleaved by sulfhydryl reagents like mercaptoethanol or OTT (2). However, many proteins that contain disulfide bridges essential for their function, for example, antibodies, cannot be recovered in a functional state by elution with sulfhydryl reagents. Thus, our idea was to use light for elution of material bound to an affinity matrix. In this report we present an efficient method of affinity purification based on the biotin-streptavidin system and elution by irradiation with light as the essential step.

Biophys. 106, 1-5. Chaiet, L. , Tausig, F. and Wolf, F. J. (1963) Antimicrob. Ag. Chemother. 3, 28-32. Sano, T. R. ( 1990) 1. Bioi. Chem. 265, 3369-3373. A. and Wilchek, M. (1988) Biochem. 1. 250, 291-294. , Rahway, NJ, p. 192. H. and Hull, R. (1986) Nuc!. Acid Res. 14, 9965-9976. M. B. (1985) 1. Chromo330, 153-158. J. F. (1977) Anal. Biochem. 81, 442-446. B. A. (1970) Meth. Enzymol. 18A,418-424. , Criscimagna, N. and Horowitz, P. (1985) Anal. Biochem. 151, 178-181. M. (1965) Biochem. 1. 94. 23c-24c.

13. O. Biotinylated nucleic acid probes prepared in this manner can be stored at -20°C for at least a year. These biotinylated probes can be used in hybridization techniques in the same manner as biotinylated probes prepared by other methods. 9 if higher concentrations of avidin are used in the reagent's preparation. Dilutions must be accounted for during calculations. Substantial decreases in the final pH of the solution caused by the use of highly buffered acidic solutions will cause a decrease in the extinction of the avidin-HABA reagent and result in underestimation of the biotin concentration of the sample.

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