Endogenous biotin-binding proteins: an overlooked factor causing false positives in streptavidin-based protein detection.
Bottom Line: Surprisingly, this protein of 125 kDa could not be purified using a ConA affinity column.Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin-associated proteins.To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.
Affiliation: Department of Bioscience Engineering, Research Group Environmental Ecology and Applied Microbiology, University of Antwerp, Antwerp, Belgium; Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, Leuven, Belgium.Show MeSH
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Mentions: To circumvent the occurrence of false positive bands completely, we suggest the digoxigenin–anti-digoxigenin (DIG–anti-DIG) detection method as a good alternative. As digoxigenin is a steroid only produced by Digitalis plants, interference of endogenous material in other species is not an issue (Chevalier et al., 1997) (cf. Fig. 2B, for a confirmation in L. rhamnosus GG). The DIG label is recognized by an anti-DIG antibody that binds with a high specificity to the small steroid molecule (only 390 Da). In practical set-ups, only the Fab fragments of the anti-DIG antibody are used to avoid (albeit rare) cross reaction with structurally related steroids (Kessler, 1991). An N-hydroxysuccinimide ester derivative with a 6-aminocaproate spacer is commercially available to label probes with DIG. Results for the L. rhamnosus GG wild type exoproteome probed with a DIG-labelled lectin mix show less bands and most importantly, at 125 kDa, no band is visible, which leads to the presumption that false positives hits are lacking (Fig. 2A). This hypothesis is confirmed by a Western blot developed using only the anti-DIG antibody (Fig. 2B).
Affiliation: Department of Bioscience Engineering, Research Group Environmental Ecology and Applied Microbiology, University of Antwerp, Antwerp, Belgium; Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, Leuven, Belgium.