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Formation of reactive oxygen species in spermatozoa of infertile patients

Immunoprecipitation To enrich PDHc-E2 or OGDHc-E2 for proteomics analysis, 5 10 6 cells were lysed in 1.5 ml IP buffer (50 mM HEPES (pH 7.0), 150 mM NaCl, 1% (vol/vol) IGEPAL CA-630, 1 mM phenylmethylsulfonyl fluoride, 10 mM Tris(2-carboxyethyl)phosphine hydrochloride, 100 mM nicotinamide, 1 Roche Complete Protease Inhibitor Cocktail) and rotated at 4 C for 30 min

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The cells were incubated continuously, with half of the medium replaced with differentiation medium every day for 3 days