Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases, which play a pivotal role in the energetic metabolism. At the present, nine isoforms are known. AKs catalyze the following reversible reaction: ATP + AMP ↔ 2 ADP, even though isoform 3 uses GTP instead ATP. For many years, the activity of AKs has been detected only after native polyacrylamide gel separations, i.e. in the absence of sodium dodecyl sulfate or methanol. In this work, we report the possibility to detect the activity of the isoforms able to use ATP as substrate, directly onto gel or nitrocellulose sheets, after denaturing SDS-PAGE and electroblotting. This method is innovative because it allows to determine simultaneously the activity and the molecular weight of AKs, especially onto nitrocellulose where bands are sharper, thanks to absence of protein diffusion.

Simultaneous detection of activity and relative molecular mass of adenylate kinases after SDS-PAGE and blotting

Ravera, Silvia;Panfoli, Isabella
2017

Abstract

Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases, which play a pivotal role in the energetic metabolism. At the present, nine isoforms are known. AKs catalyze the following reversible reaction: ATP + AMP ↔ 2 ADP, even though isoform 3 uses GTP instead ATP. For many years, the activity of AKs has been detected only after native polyacrylamide gel separations, i.e. in the absence of sodium dodecyl sulfate or methanol. In this work, we report the possibility to detect the activity of the isoforms able to use ATP as substrate, directly onto gel or nitrocellulose sheets, after denaturing SDS-PAGE and electroblotting. This method is innovative because it allows to determine simultaneously the activity and the molecular weight of AKs, especially onto nitrocellulose where bands are sharper, thanks to absence of protein diffusion.
978-1-4939-7109-1
978-1-4939-7111-4
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11567/897250
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