A Cost-Effective Immobilization Method for MBP Fusion Proteins on Microtiter Plates Using a Gelatinized Starch–Agarose Mixture and Its Application for Convenient Protein–Protein Interaction Analysis

Methods and Protocols

article

Yuri Emoto¹  Ryoya Katayama²  Emi Hibino¹  Sho Ishihara¹  Natsuko Goda¹  Takeshi Tenno¹  Yoshihiro Kobashigawa⁶  Hiroshi Morioka⁶  Hidekazu Hiroaki¹ 

[1]Laboratory of Structural Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University

[2]Division of Biological Sciences, Graduate School of Science, Nagoya University

[3]Graduate Program of Transformative Chem-Bio Research, Nagoya University

[4]WISE Program, Convolution of Informatics and Biomedical Sciences on Glocal Alliances, Nagoya University

[5]BeCellBar LLC.

[6]Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University

[7]5-1 Oe-honmachi

[8]Center for One Medicine Innovative Translational Research ,(COMIT), Nagoya University

关键词: gelatinized starch;    maltose-binding protein;    microplate-based assay;    protein–protein interaction;    dissociation constant determination;   

DOI  :  10.3390/mps6030044

学科分类：电力

来源: mdpi

【 摘 要 】

The detection and quantification of protein–protein interactions (PPIs) is a crucial technique that often involves the use of recombinant proteins with fusion protein tags, such as maltose-binding protein (MBP) and glutathione-S-transferase (GST). In this study, we improved the cohesive and sticky properties of gelatinized starch by supplementing it with agarose, resulting in a harder gel that could coat the bottom of a microtiter plate. The resulting gelatinized starch/agarose mixture allowed for the efficient immobilization of MBP-tagged proteins on the coated plates, enabling the use of indirect ELISA-like PPI assays. By using the enzymatic activity of GST as an indicator, we succeeded in determining the dissociation constants between MBP-tagged and GST-tagged proteins on 96-well microtiter plates and a microplate reader without any expensive specialized equipment.

【 授权许可】

CC BY