Fig. 4.

Investigation of TEX101 analyte identity in seminal plasma (SP) and the impact of SP pretreatment. a Size-exclusion chromatography was used to investigate TEX101 association with protein complexes and seminal microvesicles (SMVs) in the pre-vasectomy SP. Using molecular weight standards, we estimated the molecular weight of free soluble TEX101 as 28 kDa. No major complexes with other proteins were detected in the range 70–210 kDa. Some TEX101 (~14%), however, was associated with the high-molecular weight structures (>500 kDa) and found in the void volume. Ultracentifugation at 120,000 × g of the same pre-vasectomy SP pool followed by size-exclusion chromatography resulted in the non-detectable TEX101 in the high-molecular weight fractions. b The effect of guanidinium chloride (GndCl)- and sodium deoxycholate (DOC)-based treatments on TEX101 measurements by ELISA was estimated for SP pools of pre-vasectomy, post-vasectomy, or infertile men, as well as for their corresponding vesicle-free fractions and SMVs. Dotted lines represent the ELISA limit of detection. Both GndCl- and DOC-based treatments were efficient in the pre-vasectomy pools with high TEX101, with substantial amounts of TEX101 found associated with SMVs. GndCl-based treatment, however, was not efficient in the pool of infertility samples with low TEX101 (~100-fold lower than amounts in the pre-vasectomy pools