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Proteins, Expression, Isolation and Analysis> | Yeast samples were resolved on 10% SDS polyacrylamide gels were transferred to a nitrocellulose membrane at 90 V for 1 h. A 5% solution of non-fat milk powder was used for blocking and the probe used for the western blot was a 1:2500 anti-histidine antibody (Genscript, Cat. No. A001865). | Get A Quote |
A yeast deletion library was screened based on NADH fluorescence using a 384-well plate assay to identify a yeast isolate lacking a previously identified cell surface oxidase exhibiting an oscillatory pattern with a period length of 25 min and resistant to the ENOX1-specific inhibitor simalikalactone D (YNOX for yeast-specific ENOX = ENOX4). The cDNA was cloned from a yeast over expression library using NADH fluorescence analyzed by Fast Fourier transform and decomposition fits. The objective was to identify and sequence an ENOX homologue in Saccharomyces cerevisiae with a 25 min rather than a 24 min period length (YNOX). The finding identified YDR005C as the yeast ENOX protein with a temperature-independent 25... More