Endogenous biotin-binding proteins: an overlooked factor causing false positives in streptavidin-based protein detection - PubMed (original) (raw)

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

Hanne L P Tytgat et al. Microb Biotechnol. 2015 Jan.

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 rhamnosus GG with biotinylated lectin probes, a strong positive band of approximately 125 kDa 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 125 kDa 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 125 kDa 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.

© 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

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Figures

Fig 1

Fig 1

Detection of glycoproteins on Western blot using biotinylated lectins and streptavidin results in false positive hits.A. Biotinylated lectin blots – The exoproteome of wild type L . rhamnosus GG and its Δ_dltD_::TcR mutant were subjected to SDS-PAGE on NuPAGE® Novex® 12% Bis-Tris gels (Life Technologies) and subsequently blotted to PVDF membranes (Life Technologies). The prestained Kaleidoscope™ ladder (Bio-Rad) was added as a molecular weight marker. The Western blots were developed using biotinylated probes, in this case lectins. In the left panel, the Western blot was probed with biotinylated ConA, which specifically binds glucose and terminal mannose. Incubation with streptavidin conjugated to alkaline phosphatase (Roche) enabled the visual detection of positive bands using NBT and BCIP. In the right panel, the blot was developed using the same principle, but initial probing was performed using a mix of biotinylated lectins: ConA (Glc, Man), GNA (Man), HHA (Man), WGA (GlcNAc), DSL (GlcNAc), UDA (GlcNAc), Nictaba (GlcNAc), RSA (Gal, GalNAc) and PNA (Gal, GalNAc). In both Western blots and for both strains, a peculiarly strong band appeared at approximately 125 kDa.B. Using a streptavidin conjugate to sample the proteome for false positive hits – The blotted exoproteome of L . rhamnosus GG and the Δ_dltD_::TcR mutant were probed directly with a streptavidin conjugate. This resulted in the appearance of several bands, among which the strong 125 kDa band. Based on this result, we suggest that the band is not caused by a glycoprotein, but is a false positive.C. PAS glycostain does not react with the 125 kDa protein – An SDS-PAGE gel of the proteome of L . rhamnosus GG was post-stained with Periodic Acid Schiff base stain (PAS, Pro-Q® Emerald 488 stain, Life Technologies), a method to specifically stain glycosylated proteins in a gel. At 125 kDa, no band could be detected, which further supports our hypothesis that the 125 kDa signal perceived on the lectin blots is the result of a false positive hit.

Fig 2

Fig 2

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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