| Chapter title |
Analysis of Ethylene Receptors: Assay for Histidine Kinase Activity
|
|---|---|
| Chapter number | 8 |
| Book title |
Ethylene Signaling
|
| Published in |
Methods in molecular biology, January 2017
|
| DOI | 10.1007/978-1-4939-6854-1_8 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-6852-7, 978-1-4939-6854-1
|
| Authors |
G. Eric Schaller, Brad M. Binder |
| Editors |
Brad M. Binder, G. Eric Schaller |
| Abstract |
The ethylene receptors of plants exist in two subfamilies. Members of subfamily-1 have functional histidine kinase domains, whereas members of subfamily-2 have diverged histidine-kinase-like domains that in some cases have been shown to exhibit Ser/Thr kinase activity. Here, we describe a method to biochemically characterize the enzymatic activity of these kinase domains in vitro. For this purpose, the histidine kinase domain of the receptors is transgenically expressed in yeast as a fusion to glutathione-S-transferase (GST) for subsequent affinity purification. Autophosphorylation activity is assessed by the use of an in vitro kinase assay with the purified protein. Acid/base stability of the incorporated phosphate can then be used as a diagnostic for whether His, Asp, or Ser/Thr/Tyr is phosphorylated. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 4 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 1 | 25% |
| Professor > Associate Professor | 1 | 25% |
| Researcher | 1 | 25% |
| Student > Master | 1 | 25% |
| Readers by discipline | Count | As % |
|---|---|---|
| Chemistry | 2 | 50% |
| Immunology and Microbiology | 1 | 25% |
| Biochemistry, Genetics and Molecular Biology | 1 | 25% |