Molecular Screening and Protein Expression Facility, The Wistar Institute, Philadelphia, PA, United States.
Molecular Screening and Protein Expression Facility, The Wistar Institute, Philadelphia, PA, United States; Medicinal Chemistry, The Wistar Institute, Philadelphia, PA, United States; Program in Molecular and Cellular Oncogenesis, The Wistar Institute, Philadelphia, PA, United States.
Methods Enzymol. 2023;681:115-153. doi: 10.1016/bs.mie.2022.08.012. Epub 2022 Oct 3.
The In-Cell Western plate-based immunofluorescence assay is a useful methodology for monitoring protein levels and provides a facile moderate through-put method for PROTAC and degrader optimization. The method is compared to other reported assays used for PROTAC development. The advantages of this method are the greater through-put compared to Western blots due to its plate-based method and the ease to transfer between cells lines. Adherent cell lines are preferred, although suspension cells can be used following recommended modifications and precautions to the protocol. This method requires a high-quality antibody that recognizes the protein epitope in its cellular context, and in general provides data similar to Western blots with higher assay through-put.
基于细胞的 Western 板免疫荧光检测法是一种监测蛋白水平的有用方法，为 PROTAC 和降解剂的优化提供了一种简单的高通量方法。该方法与其他用于 PROTAC 开发的报道方法进行了比较。与 Western blot 相比，该方法的优势在于其板载方法具有更高的通量，并且易于在细胞系之间转移。优选贴壁细胞系，尽管可以在遵守协议的建议修改和预防措施的情况下使用悬浮细胞。该方法需要识别蛋白表位在细胞环境中的高质量抗体，并且通常提供与 Western blot 相似的数据，但具有更高的测定通量。
