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    • 4. 发明申请
    • A METHOD OF DETECTING MOLECULES IN PROXIMITY TO A TARGET MOLECULE IN A SAMPLE
    • 检测样品中目标分子的分子的方法
    • WO2017040404A1
    • 2017-03-09
    • PCT/US2016/049263
    • 2016-08-29
    • THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES
    • BAR, Daniel Z.COLLINS, Francis
    • G01N33/68
    • G01N33/6845
    • Antibody-based methods of detecting of molecules interacting with a target molecule, such as a nuclear or cytoplasmic protein, in a fixed and, optionally, permeabilized cell or tissue sample. In one example, the target molecule is bound by a primary antibody, and a secondary, peroxidase-conjugated antibody, then binds to the primary antibody. The peroxidase may also be conjugated the primary antibody. Biotin tyramide is added to the sample, and the peroxidase generates short-lived intermediates resulting in biotinylation of molecules adjacent to the peroxidase. Biotinylated molecules can be isolated from the sample by affinity interaction with avidin- or streptavidin, resulting in a fraction containing biotinylated molecules that are located in the sample in proximity to the target molecule. The fraction can be analyzed by mass-spectroscopy, Western blotting, sequencing and other techniques.
    • 在固定和任选的透化细胞或组织样品中检测与靶分子例如核或细胞质蛋白相互作用的分子的基于抗体的方法。 在一个实例中,靶分子被一级抗体结合,并且次级的,过氧化物酶缀合的抗体然后与一级抗体结合。 过氧化物酶也可以与一级抗体结合。 将生物素酪胺加入到样品中,过氧化物酶产生短暂的中间体,导致与过氧化物酶相邻的分子的生物素化。 生物素化的分子可以通过与抗生物素蛋白或链霉抗生物素蛋白亲和力相互作用从样品中分离出来,导致含有生物素化分子的部分位于样品中靠近靶分子。 该分数可以通过质谱,蛋白质印迹,测序和其他技术进行分析。