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

(2015) MICROBIAL BIOTECHNOLOGY. 8(1). p.164-168
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Abstract
Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin-based detection of glycoproteins in Lactobacillus rhamnosusGG with biotinylated lectin probes, a strong positive band of approximately 125kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A (ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin-associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin-based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.
Keywords
DIGOXIGENIN, LACTOBACILLUS-RHAMNOSUS GG

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MLA
Tytgat, Hanne LP et al. “Endogenous Biotin-binding Proteins : an Overlooked Factor Causing False Positives in Streptavidin-based Protein Detection.” MICROBIAL BIOTECHNOLOGY 8.1 (2015): 164–168. Print.
APA
Tytgat, H. L., Schoofs, G., Driesen, M., Proost, P., Van Damme, E., Vanderleyden, J., & Lebeer, S. (2015). Endogenous biotin-binding proteins : an overlooked factor causing false positives in streptavidin-based protein detection. MICROBIAL BIOTECHNOLOGY, 8(1), 164–168.
Chicago author-date
Tytgat, Hanne LP, Geert Schoofs, Michele Driesen, Paul Proost, Els Van Damme, Jos Vanderleyden, and Sarah Lebeer. 2015. “Endogenous Biotin-binding Proteins : an Overlooked Factor Causing False Positives in Streptavidin-based Protein Detection.” Microbial Biotechnology 8 (1): 164–168.
Chicago author-date (all authors)
Tytgat, Hanne LP, Geert Schoofs, Michele Driesen, Paul Proost, Els Van Damme, Jos Vanderleyden, and Sarah Lebeer. 2015. “Endogenous Biotin-binding Proteins : an Overlooked Factor Causing False Positives in Streptavidin-based Protein Detection.” Microbial Biotechnology 8 (1): 164–168.
Vancouver
1.
Tytgat HL, Schoofs G, Driesen M, Proost P, Van Damme E, Vanderleyden J, et al. Endogenous biotin-binding proteins : an overlooked factor causing false positives in streptavidin-based protein detection. MICROBIAL BIOTECHNOLOGY. 2015;8(1):164–8.
IEEE
[1]
H. L. Tytgat et al., “Endogenous biotin-binding proteins : an overlooked factor causing false positives in streptavidin-based protein detection,” MICROBIAL BIOTECHNOLOGY, vol. 8, no. 1, pp. 164–168, 2015.
@article{5976335,
  abstract     = {Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin-based detection of glycoproteins in Lactobacillus rhamnosusGG with biotinylated lectin probes, a strong positive band of approximately 125kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A (ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin-associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin-based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.},
  author       = {Tytgat, Hanne LP and Schoofs, Geert and Driesen, Michele and Proost, Paul and Van Damme, Els and Vanderleyden, Jos and Lebeer, Sarah},
  issn         = {1751-7907},
  journal      = {MICROBIAL BIOTECHNOLOGY},
  keywords     = {DIGOXIGENIN,LACTOBACILLUS-RHAMNOSUS GG},
  language     = {eng},
  number       = {1},
  pages        = {164--168},
  title        = {Endogenous biotin-binding proteins : an overlooked factor causing false positives in streptavidin-based protein detection},
  url          = {http://dx.doi.org/10.1111/1751-7915.12150},
  volume       = {8},
  year         = {2015},
}

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