Dimethyl pimelimidate dmp

Before extraction, mitochondria (200 μg) isolated from control fibroblasts were chemically cross‐linked with 1 mM disuccinimidyl glutarate (DSG) (Sigma) in isolation buffer, for 2 h on ice. The reaction was stopped by adding glycine pH 8.0 at 70 mM final for 10 min on ice. Mitochondria were pelleted, rinsed once, and extracted in 200 μl of lysis buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% n‐dodecyl‐D‐maltoside (DDM) (Sigma), and complete protease inhibitors (Roche)) on ice for 30 min. The extract was centrifuged at 20,000 × g at 4°C for 20 min, and the supernatant was pre‐cleared overnight with non‐coated Dynabeads Protein A (Invitrogen) to reduce non‐specific protein binding to the beads. Binding of indicated antibodies to Dynabeads Protein A (Invitrogen) was performed overnight. Antibodies were then cross‐linked to the beads using 20 mM dimethyl pimelimidate (DMP) (Sigma). The immunoprecipitation reaction was performed overnight at 4°C. Samples were eluted using 0.1 M glycine pH 2.5/0.5% DDM, trichloroacetic acid precipitated, and analyzed by mass spectrometry on an Orbitrap (Thermo Scientific) at the Institute de Recherches Cliniques de Montreal. The false discovery rate is < 5% with a Mascot score of 50.

Transfected HEK293T cells and male germ cells were lysed in a pull‐down buffer (PB: 25 mmol/L Tris‐HCl pH 7.5, 150 mmol/L NaCl, 0.5% Triton‐X100, 1 mmol/L EDTA, 5% glycerol and PIC) and protein concentration was determined using a BCA protein assay kit (Pierce). When indicated, GST or GST fusion proteins were cross‐linked to the glutathione‐agarose beads with Dimethyl pimelimidate (DMP, Sigma‐Aldrich. Stock concentration: 13 mg/mL). Briefly, GST protein bound to the GST‐agarose were washed 2 times with 200 mmol/L Triethanolamine pH 8.9 and incubated for 1 hour with the cross‐linking solution (50 mmol/L DMP in 200 mmol/L Triethanolamine pH 8.9). Cross‐link reaction was blocked with 100 mmol/L ethanolamine pH 8.9, and the beads were washed in PBS and used for pull‐down experiments. Cell extracts (500 µg‐1 mg) were pre‐cleared on glutathione‐agarose beads for 1 hour at 4°C, then incubated under constant shaking with GST or GST fusion proteins, that were cross‐linked or not to the beads, for 2 hours at 4°C. After three washes with the PB buffer, adsorbed proteins were eluted in SDS sample buffer and analysed by Western blotting.