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
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 p...
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MDPI AG
2023-04-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/6/3/44 |
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| author | Yuri Emoto Ryoya Katayama Emi Hibino Sho Ishihara Natsuko Goda Takeshi Tenno Yoshihiro Kobashigawa Hiroshi Morioka Hidekazu Hiroaki |
| author_facet | Yuri Emoto Ryoya Katayama Emi Hibino Sho Ishihara Natsuko Goda Takeshi Tenno Yoshihiro Kobashigawa Hiroshi Morioka Hidekazu Hiroaki |
| author_sort | Yuri Emoto |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-11T02:06:50Z |
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| id | doaj.art-ff5d59e96c6b4305a66a0bee55119b82 |
| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-03-11T02:06:50Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
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| spelling | doaj.art-ff5d59e96c6b4305a66a0bee55119b822023-11-18T11:51:42ZengMDPI AGMethods and Protocols2409-92792023-04-01634410.3390/mps6030044A 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 AnalysisYuri Emoto0Ryoya Katayama1Emi Hibino2Sho Ishihara3Natsuko Goda4Takeshi Tenno5Yoshihiro Kobashigawa6Hiroshi Morioka7Hidekazu Hiroaki8Laboratory of Structural Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Aichi, JapanDivision of Biological Sciences, Graduate School of Science, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Aichi, JapanLaboratory of Structural Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Aichi, JapanLaboratory of Structural Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Aichi, JapanLaboratory of Structural Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Aichi, JapanLaboratory of Structural Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Aichi, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University; 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Kumamoto, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University; 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Kumamoto, JapanLaboratory of Structural Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Aichi, JapanThe 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.https://www.mdpi.com/2409-9279/6/3/44gelatinized starchmaltose-binding proteinmicroplate-based assayprotein–protein interactiondissociation constant determination |
| spellingShingle | Yuri Emoto Ryoya Katayama Emi Hibino Sho Ishihara Natsuko Goda Takeshi Tenno Yoshihiro Kobashigawa Hiroshi Morioka Hidekazu Hiroaki 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 gelatinized starch maltose-binding protein microplate-based assay protein–protein interaction dissociation constant determination |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | gelatinized starch maltose-binding protein microplate-based assay protein–protein interaction dissociation constant determination |
| url | https://www.mdpi.com/2409-9279/6/3/44 |
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