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Endogenous biotin-binding proteins: an overlooked factor causing false positives in streptavidin-based protein detection.

Tytgat HL, Schoofs G, Driesen M, Proost P, Van Damme EJ, Vanderleyden J, Lebeer S - Microb Biotechnol (2014)

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.

View Article: PubMed Central - PubMed

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.

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The digoxigenin–anti-digoxigenin detection as an alternative to avoid false positive hits caused by proteins binding endogenous biotin.A. DIG-labelled lectin blots – The wild type exoproteome of L. rhamnosus GG was Western blotted and developed using a mix of DIG-labelled lectins: ConA (Glc, Man), GNA (Man), HHA (Man), WGA (GlcNAc), DSL (GlcNAc), UDA (GlcNAc), Nictaba (GlcNAc), RSA (Gal, GalNAc) and PNA (Gal, GalNAc). These lectins were labelled using digoxigenin-3-O-methyl-ε-aminocaproix acid-N-hydroxysuccinimide ester (Roche). Anti-DIG Fab antibody fragments (Roche) were used to detect proteins that reacted positively with the lectin probes. Here, we clearly see that the false positive band at 125 kDa is absent.B. Negative control with anti-DIG – Direct application of the anti-DIG Fab antibody fragments (Roche) to the Western blotted proteome of L. rhamnosus GG results in a blot on which no bands can be perceived. This confirms that the DIG–anti-DIG detection method is a good alternative for the biotin–streptavidin system, without causing false positive hits.
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fig02: The digoxigenin–anti-digoxigenin detection as an alternative to avoid false positive hits caused by proteins binding endogenous biotin.A. DIG-labelled lectin blots – The wild type exoproteome of L. rhamnosus GG was Western blotted and developed using a mix of DIG-labelled lectins: ConA (Glc, Man), GNA (Man), HHA (Man), WGA (GlcNAc), DSL (GlcNAc), UDA (GlcNAc), Nictaba (GlcNAc), RSA (Gal, GalNAc) and PNA (Gal, GalNAc). These lectins were labelled using digoxigenin-3-O-methyl-ε-aminocaproix acid-N-hydroxysuccinimide ester (Roche). Anti-DIG Fab antibody fragments (Roche) were used to detect proteins that reacted positively with the lectin probes. Here, we clearly see that the false positive band at 125 kDa is absent.B. Negative control with anti-DIG – Direct application of the anti-DIG Fab antibody fragments (Roche) to the Western blotted proteome of L. rhamnosus GG results in a blot on which no bands can be perceived. This confirms that the DIG–anti-DIG detection method is a good alternative for the biotin–streptavidin system, without causing false positive hits.

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).


Endogenous biotin-binding proteins: an overlooked factor causing false positives in streptavidin-based protein detection.

Tytgat HL, Schoofs G, Driesen M, Proost P, Van Damme EJ, Vanderleyden J, Lebeer S - Microb Biotechnol (2014)

The digoxigenin–anti-digoxigenin detection as an alternative to avoid false positive hits caused by proteins binding endogenous biotin.A. DIG-labelled lectin blots – The wild type exoproteome of L. rhamnosus GG was Western blotted and developed using a mix of DIG-labelled lectins: ConA (Glc, Man), GNA (Man), HHA (Man), WGA (GlcNAc), DSL (GlcNAc), UDA (GlcNAc), Nictaba (GlcNAc), RSA (Gal, GalNAc) and PNA (Gal, GalNAc). These lectins were labelled using digoxigenin-3-O-methyl-ε-aminocaproix acid-N-hydroxysuccinimide ester (Roche). Anti-DIG Fab antibody fragments (Roche) were used to detect proteins that reacted positively with the lectin probes. Here, we clearly see that the false positive band at 125 kDa is absent.B. Negative control with anti-DIG – Direct application of the anti-DIG Fab antibody fragments (Roche) to the Western blotted proteome of L. rhamnosus GG results in a blot on which no bands can be perceived. This confirms that the DIG–anti-DIG detection method is a good alternative for the biotin–streptavidin system, without causing false positive hits.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321382&req=5

fig02: The digoxigenin–anti-digoxigenin detection as an alternative to avoid false positive hits caused by proteins binding endogenous biotin.A. DIG-labelled lectin blots – The wild type exoproteome of L. rhamnosus GG was Western blotted and developed using a mix of DIG-labelled lectins: ConA (Glc, Man), GNA (Man), HHA (Man), WGA (GlcNAc), DSL (GlcNAc), UDA (GlcNAc), Nictaba (GlcNAc), RSA (Gal, GalNAc) and PNA (Gal, GalNAc). These lectins were labelled using digoxigenin-3-O-methyl-ε-aminocaproix acid-N-hydroxysuccinimide ester (Roche). Anti-DIG Fab antibody fragments (Roche) were used to detect proteins that reacted positively with the lectin probes. Here, we clearly see that the false positive band at 125 kDa is absent.B. Negative control with anti-DIG – Direct application of the anti-DIG Fab antibody fragments (Roche) to the Western blotted proteome of L. rhamnosus GG results in a blot on which no bands can be perceived. This confirms that the DIG–anti-DIG detection method is a good alternative for the biotin–streptavidin system, without causing false positive hits.
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).

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus