Native Agarose Gels and Contact Blotting as Means to Optimize the Protocols for the Formation of Antigen–Ligand Complexes

Abstract

Background: Protein complexes provide valuable biological information, but can be difficult to handle. Therefore, technical advancements designed to improve their manipulation are always useful. Methods: 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. Results: 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. Conclusions: 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.

Keywords

• protein complexes
• adhirons
• agarose native gels
• fluorescent immunoreagents