NDLI: Quantification of Protein Kinase A (PKA) Activity by an <i>in vitro</i> Radioactive Assay Using the Mouse Sperm Derived Enzyme.

Please wait, while we are loading the content...

Quantification of Protein Kinase A (PKA) Activity by an in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme.

Content Provider	Europe PMC
Author	Stival, Cintia Graf, Carolina Baro Visconti, Pablo E. Krapf, Dario
Copyright Year	2020
Abstract	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.
Related Links	https://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC7842637&blobtype=pdf
Volume Number	10
PubMed Central reference number	PMC7842637
Issue Number	12
PubMed reference number	33659328
Journal	Bio-protocol [Bio Protoc]
e-ISSN	23318325
DOI	10.21769/bioprotoc.3658
Language	English
Publisher	Bio-Protocol
Publisher Date	2020-06-20
Publisher Place	1075 Lorne Way, Sunnyvale, CA 94087, USA
Access Restriction	Open
Rights License	Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.
Subject Keyword	Protein Kinase A (PKA) Capacitation Sperm Kinase activity assay Fertilization
Content Type	Text
Resource Type	Article
Subject	Immunology and Microbiology Neuroscience Plant Science Biochemistry, Genetics and Molecular Biology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in