Reference - Detail
| RRC ID | 67727 |
|---|---|
| Author | Shigeto H, Ikeda T, Kuroda A, Funabashi H. |
| Title | A BRET-based homogeneous insulin assay using interacting domains in the primary binding site of the insulin receptor. |
| Journal | Anal Chem |
| Abstract |
A new homogeneous insulin assay requiring no chemical modification of an insulin recognition domain, which can be applied to continuous monitoring of the time-dependent cellular response in vitro, was developed. The carboxy-terminal α-chain (αCT) segment and first leucine-rich-repeat (L1) domain in the primary binding site on the insulin receptor were genetically fused with a bioluminescent protein (Nanoluc, Nluc) and a fluorescent protein (yellow fluorescent protein, YPet) to produce the insulin-sensing probe proteins Nluc-αCT and L1-YPet. The BRET signal was observed on simple mixing of insulin with these protein probes, in a so-called homogeneous assay. The BRET signal was proportional to the insulin concentration, and the lower detection limit was 0.8 μM. Time-dependent insulin secretion from drug-stimulated MIN6 cells was also successfully monitored continuously with the probe proteins. This BRET-based homogeneous insulin assay method is thus expected to be applicable to drug development by high-throughput screening. |
| Volume | 87(5) |
| Pages | 2764-70 |
| Published | 2015-3-3 |
| DOI | 10.1021/ac504063x |
| PMID | 25655236 |
| MeSH | Animals Bacterial Proteins / metabolism* Binding Sites Biological Assay / methods* CHO Cells Cricetinae Cricetulus Energy Transfer Glucose / pharmacology Humans Insulin / analysis* Insulin / metabolism Insulin Secretion Insulin-Secreting Cells / cytology Insulin-Secreting Cells / drug effects Insulin-Secreting Cells / metabolism* Luminescent Measurements / methods* Luminescent Proteins / metabolism* Mice Protein Binding Receptor, Insulin / chemistry Receptor, Insulin / metabolism* |
| IF | 6.785 |
| Altmetric score |
オルトメトリクス指標項目
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| The most frequently cited source | Youtube |
| Total number of mentions | 4 |
| Altmetric score changes over past 6months | 0.0 |
| Resource | |
| Human and Animal Cells | CHO-K1(RCB0285) |