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A Cost-Effective Immobilization Method of MBP-Fusion Proteins on Microtiter Plates Using a Gelatinized Starch-Agarose Mixture and Its Application for Convenient Protein–Protein Interaction Analysis
Emoto, Y.; Katayama, R.; Hibino, E.; Ishihara, S.; Goda, N.; Tenno, T.; Kobashigawa, Y.; Morioka, H.; Hiroaki, H. 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 Protoc.2023, 6, 44.
Emoto, Y.; Katayama, R.; Hibino, E.; Ishihara, S.; Goda, N.; Tenno, T.; Kobashigawa, Y.; Morioka, H.; Hiroaki, H. 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 Protoc. 2023, 6, 44.
Emoto, Y.; Katayama, R.; Hibino, E.; Ishihara, S.; Goda, N.; Tenno, T.; Kobashigawa, Y.; Morioka, H.; Hiroaki, H. 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 Protoc.2023, 6, 44.
Emoto, Y.; Katayama, R.; Hibino, E.; Ishihara, S.; Goda, N.; Tenno, T.; Kobashigawa, Y.; Morioka, H.; Hiroaki, H. 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 Protoc. 2023, 6, 44.
Abstract
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.
Chemistry and Materials Science, Physical Chemistry
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
