D'Ercole Claudia, de Marco Ario
Laboratory of Environmental and Life Sciences, University of Nova Gorica, Vipavska Cesta 13, P.O. Box 301, SI-5000 Nova Gorica, Slovenia.
Bioengineering (Basel). 2023 Sep 22;10(10):1111. doi: 10.3390/bioengineering10101111.
Protein complexes provide valuable biological information, but can be difficult to handle. Therefore, technical advancements designed to improve their manipulation are always useful.
We investigated the opportunity to exploit native agarose gels and the contact blot method for the transfer of native proteins to membranes as means for optimizing the conditions for obtaining stable complexes. As a simple model of protein-protein interactions, an antigen-ligand complex was used in which both proteins were fused to reporters.
At each step, it was possible to visualize both the antigen, fused to a fluorescent protein, and the ligand, fused to a monomeric ascorbate peroxidase (APEX) and, as such, a way to tune the protocol. The conditions for the complex formation were adapted by modifying the buffer conditions, the concentration of the proteins and of the cross-linkers.
The procedure is rapid, inexpensive, and the several detection opportunities allow for both the monitoring of complex stability and the preservation of the functionality of its components, which is critical for understanding their biomedical implications and supporting drug discovery. The overall protocol represents a handy alternative to gel filtration, uses very standard and ubiquitous equipment, and can be implemented rapidly and without specific training.
蛋白质复合物提供了有价值的生物学信息，但处理起来可能很困难。因此，旨在改进其操作的技术进步总是有用的。
我们研究了利用天然琼脂糖凝胶和接触印迹法将天然蛋白质转移到膜上的机会，以此作为优化获得稳定复合物条件的手段。作为蛋白质-蛋白质相互作用的一个简单模型，使用了一种抗原-配体复合物，其中两种蛋白质都与报告基因融合。
在每个步骤中，都可以同时可视化与荧光蛋白融合的抗原和与单体抗坏血酸过氧化物酶（APEX）融合的配体，从而找到一种调整实验方案的方法。通过改变缓冲液条件、蛋白质浓度和交联剂浓度来调整复合物形成的条件。
该方法快速、廉价，多种检测机会既能监测复合物的稳定性，又能保持其组分的功能，这对于理解其生物医学意义和支持药物发现至关重要。整个方案是凝胶过滤的便捷替代方法，使用非常标准且普遍的设备，无需特定培训即可快速实施。
