| Autor: |
Cintia Stival, Carolina Baro Graf, Pablo Visconti, Dario Krapf |
| Jazyk: |
angličtina |
| Rok vydání: |
2020 |
| Předmět: |
|
| Zdroj: |
Bio-Protocol, Vol 10, Iss 12 (2020) |
| Druh dokumentu: |
article |
| ISSN: |
2331-8325 |
| DOI: |
10.21769/BioProtoc.3658 |
| Popis: |
In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique faces two main disadvantages: it is not a direct measure of the kinase activity and it is a time-consuming approach. However, since PKA can be readily obtained from sperm extracts, in vitro assays such as the “radioactive assay” can be performed using the native enzyme. Unlike western-blot, the radioactive assay is a straightforward technique to evaluate PKA activity by quantification of incorporated 32P into a peptidic substrate. This approach easily allows the analysis of different agonists or antagonists of PKA. Since mouse sperm is a rich source of soluble PKA, this assay allows a simple fractionation that renders PKA usable both for in vitro testing of drugs on PKA activity and for following changes of PKA activity during the onset of capacitation. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
|
