Identification of RSVP14 and RSVP20 components by two-dimensional electrophoresis and Western-blotting

Reproduction in Domestic Animals = Zuchthygiene
J A CardozoTeresa Muiño-Blanco

Abstract

We have already shown that RSVP14 and RSVP20, two ram seminal plasma (SP) proteins postulated to be involved in sperm capacitation and gamete interaction can protect spermatozoa against cold-shock. In this study, we use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the analysis of SP proteins of Rasa Aragonesa rams, using enhanced protein solubilization in the presence of tributyl phosphine (TBP) and a polyacrylamide linear gradient gel with a narrow pH range (4-7). The image analysis of the 2D map detected 195 protein spots, with isoelectric points (pIs) ranging from 4.5 to 6.6, and molecular weight (M(r)) from 11.7 to 90.4. Staining of 2D gels with Pro-Q Emerald 300 Glycoprotein Stain revealed that most significant proteins in ram SP are glycosylated. The removing of protein N-linked oligosaccharides improved the gel resolution. 2D-PAGE analysis of the whole fraction 6 (F6) separated from ram SP by exclusion chromatography showed six main protein spots, four (a, b, c, d) in the 14 kDa and two (e, f) in the 20 kDa region. Western-blot analyses indicated that the anti-P14 antibody recognized four spots on the SP map, 4, 5, 6 and 7, that matched with spots a, b, c, d of F6 map. The anti-P20 antibody recognized...Continue Reading

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Citations

Jun 5, 2012·Journal of Proteomics·Carlos Eduardo A SouzaArlindo A Moura
Jun 12, 2013·Journal of Proteomics·X DruartS P de Graaf
Apr 17, 2007·Theriogenology·E MartiJ A Cebrián-Pérez
Mar 2, 2021·Frontiers in Veterinary Science·Eduardo G AisenTeodosio Huanca Mamani

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