Protein extraction and western blot experiments on whole testes and elutriated spermatids were performed as described in Comptour et al.65 (link) In brief, 15 μl of each immunoprecipitated sample and a volume of input sample corresponding to 10% of IP sample were denatured using 4 × NuPAGE LDS sample buffer (Life Technologies) with 10% β – mercaptoethanol, boiled for 10 min at 95 °C and loaded. Extraction of sperm proteins was as follow: five millions of spermatozoa (purity ⩾99%) were resuspended in 200 μl of 4 × NuPAGE LDS sample buffer (Life Technologies) and boiled for 10 min at 95 °C. Ten microliter of sample were loaded per lane. Antibody against SLY14 (link) was diluted 1/3000, antibody against SLX/SLXL1,69 (link) 1/6000, anti-H3K79me2 (ab-3594 from Abcam), anti-TBLX1R1 (ab 24550 from Abcam), anti-Tubulin antibody (T-9026 from Sigma-Aldrich), anti-panH3 antibody (05-928 from Abcam), anti-FLAG (M5 from Sigma-Aldrich) and anti-Hup2B antibody (Briar Patch Biosciences, Grass Valley, CA, USA), 1/1000.
Protocol detail
Protein Extraction and Western Blot Analysis
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Anti antibody
Antibody
Buffer
Grass
Mercaptoethanol
Protein
Sperm proteins
Spermatids
Spermatozoa
Testes
Tubulin
Western blot
Corresponding Organization :
Other organizations : Institut Cochin, Sorbonne Paris Cité, Université Paris Cité, Délégation Paris 5, Sorbonne Université, Australian National University, Maternité Port Royal, Centre Hospitalier Sainte-Anne, Institut des Maladies Génétiques Imagine, Assistance Publique – Hôpitaux de Paris, Genetique Reproduction and Developpement
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- Protein extraction and western blot experiments on whole testes and elutriated spermatids
- Protein expression of SLY, SLX/SLXL1, H3K79me2, TBLX1R1, Tubulin, panH3, FLAG, and Hup2B
- Volume of input sample corresponding to 10% of IP sample
- Spermatozoa purity ⩾99%
- Dilution of antibodies: SLY 1/3000, SLX/SLXL1 1/6000, anti-H3K79me2 1/1000, anti-TBLX1R1 1/1000, anti-Tubulin 1/1000, anti-panH3 1/1000, anti-FLAG 1/1000, anti-Hup2B 1/1000
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