A native, affinity‐based protein blot for the analysis of streptavidin heterogeneity: Consequences for the specificity of streptavidin mediated binding assays
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Titel:
A native, affinity‐based protein blot for the analysis of streptavidin heterogeneity: Consequences for the specificity of streptavidin mediated binding assays
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<jats:title>Abstract</jats:title><jats:p>Commercial preparations of streptavidin, a bacterial biotin‐binding protein, were analyzed by isoelectric focusing combined with an affinity‐based protein blot using biotinylated, protein‐saturated nitrocellulose. The colorimetrical detection of strep‐tavidin with biotinylated alkaline phosphatase allows the selective visualization of streptavidin molecules with at least two active biotin‐binding sites. Dependent on the preparation, seven to sixteen streptavidin forms were found with isoelectric points ranging from 5 to 8. Molecular weight analysis of the subunits of streptavidin showed that the observed heterogeneity was mainly due to limited proteolysis, which does not destroy the biotin‐binding activity. The preparations differed also in the nonspecific reactivity of streptavidin with single‐stranded DNA, bovine serum albumin and Tween 20. No relationship was observed between heterogeneity and non‐specific binding activity. Data obtained from protein blots onto nitrocellulose saturated with single‐stranded DNA showed that it cannot be excluded that streptavidin with only a single active biotin‐binding site is mainly responsible for the nonspecific reactivity of some streptavidin preparations.</jats:p>