Na Ayutthaya Pratchaya Pramoj, Lundberg Derek, Weigel Detlef, Li Lei
Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.
Curr Protoc Plant Biol. 2020 Jun;5(2):e20107. doi: 10.1002/cppb.20107.
Protein-protein interactions, including oligomerization, are involved in regulation of many cellular processes. Unfortunately, many proteins are expressed at a very low level in vivo, making it challenging to observe oligomerization by size-exclusion chromatography, also known as gel filtration. In this protocol, we present detailed steps to perform blue native polyacrylamide gel electrophoresis (BN-PAGE), a method to study protein oligomers in plants. The article describes protein sample preparation from transgenic Arabidopsis thaliana and running a BN-PAGE gel followed by direct western blotting or, alternatively, two-dimensional sodium dodecyl sulfide-polyacrylamide gel electrophoresis (2D SDS-PAGE). This protocol will be helpful for new researchers conducting related experiments to analyze stable protein interactions including homo- or hetero-oligomerization in plants. © 2020 The Authors.
蛋白质-蛋白质相互作用，包括寡聚化，参与许多细胞过程的调控。不幸的是，许多蛋白质在体内的表达水平非常低，这使得通过尺寸排阻色谱法（也称为凝胶过滤法）观察寡聚化具有挑战性。在本方案中，我们介绍了进行蓝色天然聚丙烯酰胺凝胶电泳（BN-PAGE）的详细步骤，这是一种研究植物中蛋白质寡聚体的方法。本文描述了从转基因拟南芥中制备蛋白质样品，并进行BN-PAGE凝胶电泳，随后直接进行蛋白质免疫印迹，或者进行二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（2D SDS-PAGE）。该方案将有助于新的研究人员进行相关实验，以分析植物中包括同型或异型寡聚化在内的稳定蛋白质相互作用。© 2020作者。
