Stewart Adam, Banerji Udai
The Institute of Cancer Research, London, UK.
The Royal Marsden, Sycamore House, Downs Road, Sutton, London, SM2 5PT, UK.
Methods Mol Biol. 2017;1636:119-131. doi: 10.1007/978-1-4939-7154-1_9.
The study of protein phosphorylation is critical for the advancement of our understanding of cellular responses to external and internal stimuli. Phosphorylation, the addition of phosphate groups, most often occurs on serine, threonine, or tyrosine residues due to the action of protein kinases. This structural change causes the protein to become activated (or deactivated) and enables it in turn to initiate the phosphorylation of other proteins in a cascade, eventually causing cell-wide changes such as apoptosis, cell differentiation, and growth (among others). Cellular phosphoprotein pathway dysregulation by mutation or chromosomal instability can often give the cell a selective advantage and lead to cancer. Obviously the understanding of these systems is of huge importance to the field of oncology.This chapter aims to provide a "how to" manual for one such technology, the 96-well plate-based xMAP platform from Luminex. The system utilizes antibody-bound free-floating magnetic spheres which can easily be removed from suspension via magnetization. There are 100 unique bead sets (moving up to 500 bead sets for the most recent system) identified by the ratio of two dyes coating the microsphere. Each bead set is conjugated to a specific antibody which allows targeted protein extraction from low-concentration lysate solution. Biotinylated secondary antibodies/streptavidin-R-phycoerythrin (SAPE) complexes provide the quantification mechanism for the phosphoprotein of interest.
蛋白质磷酸化的研究对于我们深入理解细胞对外部和内部刺激的反应至关重要。磷酸化，即磷酸基团的添加，由于蛋白激酶的作用，最常发生在丝氨酸、苏氨酸或酪氨酸残基上。这种结构变化会使蛋白质被激活（或失活），进而使其能够启动其他蛋白质的磷酸化级联反应，最终导致全细胞范围的变化，如细胞凋亡、细胞分化和生长（等等）。由于突变或染色体不稳定导致的细胞磷蛋白途径失调通常会赋予细胞选择性优势并导致癌症。显然，对这些系统的理解在肿瘤学领域具有极其重要的意义。本章旨在为一种此类技术提供一份“操作指南”，即来自Luminex的基于96孔板的xxxuminex。该系统利用与抗体结合的游离磁性微球，这些微球可通过磁化轻松从悬浮液中去除。通过包被微球的两种染料的比例可识别出100种独特的微球组（最新系统可达500种微球组）。每个微球组都与一种特定抗体偶联，从而能够从低浓度裂解液中靶向提取蛋白质。生物素化二抗/链霉亲和素 - R - 藻红蛋白（SAPE）复合物为目标磷蛋白提供定量机制。
