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[求助]
怎么检测偶联在磁珠上的生物素标记的DNA个数？求大神解答 已有1人参与
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| 我有一个链霉亲和素的磁珠，偶联生物素标记的DNA后，有什么方法可以检测每个磁珠上带DNA的个数? 求各位大神帮忙，非常感谢！ |
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西门吹雪170: 金币+1, 鼓励回帖交流 2017-05-18 07:41:09
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Just follow this one but change to measure the biotin-oligo in the supernatant after coupling to the beads. Bayer, E. A., et al. (1986). "A sensitive enzyme assay for biotin, avidin, and streptavidin." Analytical Biochemistry 154(1): 367-370. Reciprocal enzyme assays are described for the vitamin biotin and for the biotin-binding proteins avidin and streptavidin. The assays are based on the following steps: (a) biotinylated bovine serum albumin is adsorbed onto microtiter plates; (b) streptavidin (or avidin) is bound to the biotincoated plates: (c) biotinylated enzyme (in this case alkaline phosphatase) is then interacted with the free biotin-binding sites on the immobilized protein. For biotin assay, competition between the free vitamin and the biotinylated enzyme is carried out between steps (b) and (c). The method takes advantage of the four biotin-binding sites which characterize both avidin and streptavidin. The method is extremely versatile and accurate over a concentration range exceeding three orders of magnitude. The lower limits of detection are approximately 2 pg/ml (0.2 pg/sample) for biotin and less than 100 ng/ml (10 ng/sample) for either avidin or streptavidin. |
4楼2017-05-18 01:57:09
5楼2017-06-06 14:34:46
