title: A native, affinity-based protein blot for the analysis of streptavidin heterogeneity: consequences for the specificity of streptavidin mediated binding assays.
abstract: 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 streptavidin 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.
authors.affiliation: Physiologisches Institut der Universität Zürich-Irchel, Switzerland.
authors.forename: J
authors.initials: J
authors.lastname: Dittmer
authors.forename: A
authors.initials: A
authors.lastname: Dittmer
authors.forename: R
authors.initials: R
authors.lastname: Della Bruna
authors.forename: V
authors.initials: V
authors.lastname: Kasche
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000493:pharmacokinetics; D001710:Biotin; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D015151:Immunoblotting / Q000379:methods*; D007525:Isoelectric Focusing; D012710:Serum Albumin, Bovine; D019809:Streptavidin
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D012710:Serum Albumin, Bovine; D001710:Biotin; D019809:Streptavidin