PreprintArticleVersion 1Preserved in Portico This version is not peer-reviewed
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
Version 1
: Received: 20 February 2023 / Approved: 22 February 2023 / Online: 22 February 2023 (14:53:42 CET)
How to cite:
Emoto, Y.; Katayama, R.; Hibino, E.; Ishihara, S.; Goda, N.; Tenno, T.; Kobashigawa, Y.; Morioka, H.; Hiroaki, H. 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. Preprints2023, 2023020386. https://doi.org/10.20944/preprints202302.0386.v1.
Emoto, Y.; Katayama, R.; Hibino, E.; Ishihara, S.; Goda, N.; Tenno, T.; Kobashigawa, Y.; Morioka, H.; Hiroaki, H. 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. Preprints 2023, 2023020386. https://doi.org/10.20944/preprints202302.0386.v1.
Cite as:
Emoto, Y.; Katayama, R.; Hibino, E.; Ishihara, S.; Goda, N.; Tenno, T.; Kobashigawa, Y.; Morioka, H.; Hiroaki, H. 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. Preprints2023, 2023020386. https://doi.org/10.20944/preprints202302.0386.v1.
Emoto, Y.; Katayama, R.; Hibino, E.; Ishihara, S.; Goda, N.; Tenno, T.; Kobashigawa, Y.; Morioka, H.; Hiroaki, H. 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. Preprints 2023, 2023020386. https://doi.org/10.20944/preprints202302.0386.v1.
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.
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.
