Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis.
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.
Department of Life Science, Hanyang University, Seoul 04763, Korea. chlghgh@nate.com.|Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Korea. chlghgh@nate.com.
Hoseok
H
Choi
Department of Life Science, Hanyang University, Seoul 04763, Korea. charitybomi@gmail.com.|Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Korea. charitybomi@gmail.com.
Bomi
B
Choi
Department of Urology, College of Medicine, Dankook University, Cheil General Hospital, Seoul 04619, Korea. jtandro@cgh.co.kr.
Ju Tae
JT
Seo
Department of Convergence Medicine, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea. kjlee@amc.seoul.kr.
Kyung Jin
KJ
Lee
Department of Life Science, Hanyang University, Seoul 04763, Korea. mcgye@hanyang.ac.kr.|Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Korea. mcgye@hanyang.ac.kr.
Myung Chan
MC
Gye
Department of Life Science, Hanyang University, Seoul 04763, Korea. ypilkim@hanyang.ac.kr.|Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Korea. ypilkim@hanyang.ac.kr.|Institute of Nano Science and Technology, Hanyang University, Seoul 04763, Korea. ypilkim@hanyang.ac.kr.|Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04763, Korea. ypilkim@hanyang.ac.kr.
Young-Pil
YP
Kim
D000818:Animals; D004586:Electrophoresis / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D038362:Glycogen Synthase Kinase 3 / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007248:Infertility, Male / Q000175:diagnosis* / Q000201:enzymology; D008297:Male; D051379:Mice; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D013081:Sperm Motility / Q000502:physiology; D013094:Spermatozoa / Q000201:enzymology*
D005456:Fluorescent Dyes; D010455:Peptides; D038362:Glycogen Synthase Kinase 3
GSK3; gel shift assay; isoelectric point; phosphorylation; sperm; testis