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

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 t...

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Main Authors: Claudia D’Ercole, Ario de Marco
Format: Article
Language:English
Published: MDPI AG 2023-09-01
Series:Bioengineering
Subjects:
Online Access:https://www.mdpi.com/2306-5354/10/10/1111
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author Claudia D’Ercole
Ario de Marco
author_facet Claudia D’Ercole
Ario de Marco
author_sort Claudia D’Ercole
collection DOAJ
description 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.
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spelling doaj.art-b9464b8426f042bfa23c4bc0027aa79f2023-11-19T15:41:05ZengMDPI AGBioengineering2306-53542023-09-011010111110.3390/bioengineering10101111Native Agarose Gels and Contact Blotting as Means to Optimize the Protocols for the Formation of Antigen–Ligand ComplexesClaudia D’Ercole0Ario de Marco1Laboratory of Environmental and Life Sciences, University of Nova Gorica, Vipavska Cesta 13, P.O. Box 301, SI-5000 Nova Gorica, SloveniaLaboratory of Environmental and Life Sciences, University of Nova Gorica, Vipavska Cesta 13, P.O. Box 301, SI-5000 Nova Gorica, SloveniaBackground: 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.https://www.mdpi.com/2306-5354/10/10/1111protein complexesadhironsagarose native gelsfluorescent immunoreagents
spellingShingle Claudia D’Ercole
Ario de Marco
Native Agarose Gels and Contact Blotting as Means to Optimize the Protocols for the Formation of Antigen–Ligand Complexes
Bioengineering
protein complexes
adhirons
agarose native gels
fluorescent immunoreagents
title Native Agarose Gels and Contact Blotting as Means to Optimize the Protocols for the Formation of Antigen–Ligand Complexes
title_full Native Agarose Gels and Contact Blotting as Means to Optimize the Protocols for the Formation of Antigen–Ligand Complexes
title_fullStr Native Agarose Gels and Contact Blotting as Means to Optimize the Protocols for the Formation of Antigen–Ligand Complexes
title_full_unstemmed Native Agarose Gels and Contact Blotting as Means to Optimize the Protocols for the Formation of Antigen–Ligand Complexes
title_short Native Agarose Gels and Contact Blotting as Means to Optimize the Protocols for the Formation of Antigen–Ligand Complexes
title_sort native agarose gels and contact blotting as means to optimize the protocols for the formation of antigen ligand complexes
topic protein complexes
adhirons
agarose native gels
fluorescent immunoreagents
url https://www.mdpi.com/2306-5354/10/10/1111
work_keys_str_mv AT claudiadercole nativeagarosegelsandcontactblottingasmeanstooptimizetheprotocolsfortheformationofantigenligandcomplexes
AT ariodemarco nativeagarosegelsandcontactblottingasmeanstooptimizetheprotocolsfortheformationofantigenligandcomplexes