Figure 1. Detection of S-nitrosylated proteins in human spermatozoa with biotin switch assay.

Intact human sperm were treated with GSNO, GSH, CSNO, exhausted CSNO or oxidised GSH (GSSG) or incubated with no treatment (no treatment) for 1hr at 37°C prior biotin switch assay as described in Materials and Methods. A series of controls were performed to assess the specificity of the biotin switch assay. A) Firstly where indicated, the assay was tested in the absence of biotin-HPDP to show if any endogenously biotinylated proteins could be detected. Secondly where indicated, labelling with biotin-HPDP was performed with or without ascorbate, MMTS or in presence of DTT. B) GSSG was used to assess that the assay was specific for S-nitrosylation and not S-glutathionylation. Proteins were separated by SDS-PAGE (10%) and transferred onto nitrocellulose membrane. Detection of biotinylated proteins was achieved using anti-biotin antibody. The relative masses of protein standards are shown on the left. Results of 1 experiment presented are representative of 6 others performed with different sperm donors.