Endogenous biotin-binding proteins: an overlooked factor causing false positives in streptavidin-based protein detection.
Endogenous biotin-binding proteins: an overlooked factor causing false positives in streptavidin-based protein detection.
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DOI：
10.1111/1751-7915.12150
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发表时间：
2015-01
影响因子：
5.7
通讯作者：
Lebeer S
中科院分区：
文献类型：
--
作者：
Tytgat HL;Schoofs G;Driesen M;Proost P;Van Damme EJ;Vanderleyden J;Lebeer S
生物素化广泛用于DNA、RNA和蛋白质探测测定，因为这种分子通常对其底物的生物活性没有影响。在用生物素化凝集素探针对鼠李糖乳杆菌GG中的糖蛋白进行基于链霉亲和素的检测期间，观察到存在于不同细胞级分中的约125 kDa的强阳性条带。 这种潜在的糖蛋白与伴刀豆球蛋白A（ConA）反应强烈，后者是一种特异性结合葡萄糖和甘露糖残基的凝集素。令人惊讶的是，这种125 kDa的蛋白质不能使用ConA亲和柱纯化。通过阳离子和阴离子交换色谱分离的蛋白质的Edman降解导致将条带鉴定为丙酮酸羧化酶，这是一种结合生物素作为辅因子的125 kDa的酶。仅使用链霉亲和素缀合物的检测导致蛋白质的更多假阳性信号，也在细胞外级分中，指示生物素相关蛋白质。事实上，生物素是许多羧化酶的已知辅因子。因此，当使用基于链霉亲和素的检测时，例如通过仅使用链霉亲和素缀合物进行印迹，应考虑生物素化蛋白质探针的假阳性条带的潜在发生。为了避免这些假阳性，也可以使用替代方法，如基于地高辛标记的检测。
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.
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影响因子：
6.4
作者：
Lebeer S;Claes IJ;Balog CI;Schoofs G;Verhoeven TL;Nys K;von Ossowski I;de Vos WM;Tytgat HL;Agostinis P;Palva A;Van Damme EJ;Deelder AM;De Keersmaecker SC;Wuhrer M;Vanderleyden J
通讯作者：
Vanderleyden J
影响因子：
3.9
作者：
CHAIET, L;WOLF, FJ
通讯作者：
WOLF, FJ
影响因子：
56.9
作者：
Coyne, MJ;Reinap, B;Comstock, LE
通讯作者：
Comstock, LE
影响因子：
4.4
作者：
Velez, Monica Perea;Verhoeven, Tine L. A.;De Keersmaecker, Sigrid C. J.
通讯作者：
De Keersmaecker, Sigrid C. J.
影响因子：
3.7
作者：
Chen, Tingting;Hedman, Lea;Hedman, Klaus
通讯作者：
Hedman, Klaus