Isern Jaime A, Majumdar Abhirup, Richter Annika, Fiedler Dorothea
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.
Institut für Chemie, Humboldt-Universität zu Berlin, Berlin, Germany.
Methods Mol Biol. 2025;2972:125-134. doi: 10.1007/978-1-0716-4799-8_10.
Pulldown experiments isolate molecular interactions using a "bait" molecule on solid supports, often leveraging the biotin-streptavidin system. Here, a streamlined workflow is described, which employs biotinylated inositol phosphate (InsPs) and inositol pyrophosphate (PP-InsP) probes to enrich target proteins from complex proteomes. The reagents are first immobilized onto streptavidin-coated beads, then exposed to cell lysates, and subsequently washed to remove nonspecific interactions. The enriched proteins are then eluted and analyzed via western blot or quantitative mass spectrometry. This approach leverages biotin-tagged probes to enhance coupling efficiency, simplify workflows, and enable diverse applications.
下拉实验利用固相支持物上的“诱饵”分子来分离分子间相互作用，通常借助生物素-链霉亲和素系统。在此，我们描述了一种简化的工作流程，该流程使用生物素化的肌醇磷酸（InsPs）和肌醇焦磷酸（PP-InsP）探针从复杂蛋白质组中富集靶蛋白。首先将试剂固定在链霉亲和素包被的磁珠上，然后使其与细胞裂解物接触，随后进行洗涤以去除非特异性相互作用。然后洗脱富集的蛋白质，并通过蛋白质免疫印迹或定量质谱分析。这种方法利用生物素标记的探针来提高偶联效率、简化工作流程并实现多种应用。
