Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift ElectrophoresisReportar como inadecuado




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1

Department of Life Science, Hanyang University, Seoul 04763, Korea

2

Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Korea

3

Department of Urology, College of Medicine, Dankook University, Cheil General Hospital, Seoul 04619, Korea

4

Department of Convergence Medicine, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea

5

Institute of Nano Science and Technology, Hanyang University, Seoul 04763, Korea

6

Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04763, Korea



These authors contributed equally to this work.





*

Authors to whom correspondence should be addressed.



Academic Editors: Jong Seung Kim and Min Hee Lee

Abstract Assaying the glycogen synthase kinase-3 GSK3 activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated primed peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts. View Full-Text

Keywords: gel shift assay; GSK3; testis; sperm; isoelectric point; phosphorylation gel shift assay; GSK3; testis; sperm; isoelectric point; phosphorylation





Autor: Hoseok Choi 1,2,†, Bomi Choi 1,2,†, Ju Tae Seo 3, Kyung Jin Lee 4, Myung Chan Gye 1,2,* and Young-Pil Kim 1,2,5,6,*

Fuente: http://mdpi.com/



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