Amicon ultra 0.5 100 kda centrifugal filter

The anti-Smc3 antibody used was rabbit anti-Smc3 (Abcam ab9263). The anti-Rec8 antibody was generated in-house using a previously characterized epitope [47 (link)]. The control IgG used was a normal rabbit IgG (Millipore 12-370). With the exception of anti-Smc3, all antibodies were concentrated using Amicon Ultra-0.5 100 kDa centrifugal filter devices (Millipore) to remove traces of azide and replace the buffer with PBS. Following concentrations of antibodies were used: anti-Smc3 (1 mg/ml), anti-Rec8 (2 mg/ml) and control IgG (2 mg/ml). Prior to microinjection into eggs, the antibodies were spun at 10,000 rpm (4°C) for 10 minutes and supplemented with NP-40 at a final concentration of 0.05%. Antibody microinjection into eggs was performed as described previously for mRNA microinjection [52 (link)]. For full depletion experiments in the metaphase of meiosis II, a bolus of 6 pl of anti-Smc3 or anti-Rec8 was microinjected into the eggs, whereas for partial depletion experiments 2 pl of the anti-Smc3 antibody were microinjected. For partial depletion of cohesins in meiosis I, a bolus of 4 pl of the anti-Smc3 antibody was microinjected 4.5-5.5 hours after the oocytes were released from prophase arrest. The oocytes were then fixed 7 h 15 min – 8 hours after the release.