Croop Benjamin, Han Kyu Young
CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, FL, United States of America.
Phys Biol. 2019 Mar 22;16(3):035002. doi: 10.1088/1478-3975/ab0792.
The single-molecule pull-down (SiMPull) assay analyzes molecular complexes in physiological conditions from cell or tissue lysates. Currently the approach requires a lengthy sample preparation process, which has largely prevented the widespread adoption of this technique in bioanalysis. Here, we present a simplified SiMPull assay based upon dichlorodimethylsilane-Tween-20 passivation and F(ab) fragment labeling. Our passivation is a much shorter process than the standard polyethylene glycol passivation used in most single-molecule studies. The use of F(ab) fragments for indirect fluorescent labeling rather than divalent F(ab') or whole IgG antibodies allows for the pre-incubation of the detection antibodies, reducing the sample preparation time for single-molecule immunoprecipitation samples. We examine the applicability of our approach to recombinant proteins and endogenous proteins from mammalian cell lysates.
单分子下拉（SiMPull）分析可在生理条件下分析细胞或组织裂解物中的分子复合物。目前，该方法需要冗长的样品制备过程，这在很大程度上阻碍了这项技术在生物分析中的广泛应用。在此，我们提出一种基于二氯二甲基硅烷 - 吐温20钝化和F(ab)片段标记的简化SiMPull分析方法。我们的钝化过程比大多数单分子研究中使用的标准聚乙二醇钝化过程要短得多。使用F(ab)片段进行间接荧光标记而非二价F(ab')或完整IgG抗体，使得检测抗体能够进行预孵育，从而减少了单分子免疫沉淀样品的样品制备时间。我们检验了我们的方法对来自哺乳动物细胞裂解物的重组蛋白和内源性蛋白的适用性。
