Artifactual detection of biotin on histones by streptavidin
Artifactual detection of biotin on histones by streptavidin
复制标题
DOI：
10.1016/j.ab.2007.09.003
复制
发表时间：
2008-02-01
影响因子：
2.9
通讯作者：
Polyak, S. W.
中科院分区：
文献类型：
--
作者：
Bailey, L. M.;Ivanov, R. A.;Polyak, S. W.
生物素化是最近报道的翻译后对历史的修饰。全新羧化酶合成酶（HCS）和生物素酶被认为是生物素化酶。然而，生物素转移的机制和调控的细节仍不清楚。在这里，我们报告，在细胞培养系统中，低生物素可用性减少羧化酶的生物素化，但明显的生物素化历史不受影响。尽管生物素消耗对细胞活力和增殖有不利影响。进一步分析广泛使用的检测组蛋白上生物素的方法——链霉亲和素印迹法，发现链霉亲和素与组蛋白相互作用独立于生物素结合。链霉亲和素与游离生物素的预孵育减少了与生物素化羧化酶的结合，但没有阻断与历史的结合。为了研究生物素化的历史，使用一种独立于链霉亲和素的替代检测方法，分析了14c生物素掺入生物素化蛋白的情况。放射性标记的生物素很容易在羧化酶上检测到，但在组蛋白上检测不到，这意味着与组蛋白蛋白结合的细胞核中生物素水平非常低（< 0.03%生物素化）。总之，我们警告不要使用链霉亲和素来研究历史生物素化。皇冠版权(D 2007出版的爱思唯尔公司。版权所有。
Biotinylation is a recent addition to the list of reported posttranslational modifications made to histories. Holocarboxylase synthetase (HCS) and biotinidase have been implicated as biotinylating enzymes. However, the details of the mechanism and the regulation of biotin transfer on and off histories remains unclear. Here we report that in a cell culture system low biotin availability reduces biotinylation of carboxylases, yet apparent biotinylation of histories is unaffected. This is despite biotin depletion having detrimental effects on cell viability and proliferation. Further analysis of the widely used method for detecting biotin on histones, streptavidin blotting, revealed that streptavidin interacts with histones independently of biotin binding. Preincubation of streptavidin with free biotin reduced binding to biotinylated carboxylases but did not block binding to histories. To investigate biotinylation of histories using an alternative detection method independent of streptavidin, incorporation of 14 C biotin into biotinylated proteins was analyzed. Radiolabeled biotin was readily detectable on carboxylases but not on histones, implying very low levels of biotin in the nucleus attached to histone proteins (< 0.03% biotinylation). In conclusion, we would caution against the use of streptavidin for investigating historic biotinylation. Crown copyright (D 2007 Published by Elsevier Inc. All rights reserved.