2.0 ml lb microtube

DMP cross-linked beads were gently well-mixed in 1 mL of bead wash buffer then pipetted in 100 μL aliquots (0.5 mg beads/sample) into Sarstedt 2.0 mL LB microtubes. Monkey heart tissue samples (20 mg to 200 mg) were weighed and transferred to a Sarstedt 2.0 mL LB microtube. Wash and dry the tube, accurately re-weigh it and record the amount of heart tissue. RIPA lysis buffer (500 μL) containing the protease inhibitor cocktail was added to heart tissue followed by approximately, 30–50 stainless steel beads (0.9–2.0 mm). Homogenization was conducted using the Bullet Blender Gold homogenizer (Next Advance, Troy, NY) at a speed of 10 for 5 min at 4 °C. Samples were then lysed completely by additional probe sonication on ice with 30 pulses at power 4 using a sonic dismembranator (Fisher Scientific, Pittsburgh, PA). The SILAC-hFXN-M internal standard solution (20 μL of 2 μg/mL, 40 ng) was then added to each sample and appropriate amounts of hFXN-M standards ng to 200 ng (4, 10, 15, 20, 30, 40, 80, 100, 150, 200 ng) in 5% BSA solution for preparation of the standard curve^{12 (link)}. Supernatants were removed from the DMP cross-linked beads and tissue and BSA standard samples added into the Sarstedt 2.0 mL LB microtubes containing the DMP cross-linked beads. Samples were then incubated with the beads at 4 °C overnight on the rotator.

DMP cross-linked beads were gently well-mixed in 1 mL of bead wash buffer then pipetted in 100 μL aliquots (0.5 mg beads/sample) into Sarstedt 2.0 mL LB microtubes. Monkey heart tissue samples (20 mg to 200 mg) were weighed and transferred to a Sarstedt 2.0 mL LB microtube. The tube was washed and dried, accurately re-weighed, and the amount of heart tissue recorded. RIPA lysis buffer (500 μL) containing the protease inhibitor cocktail was added to heart tissue followed by approximately, 30–50 stainless steel beads (0.9–2.0 mm). Homogenization was conducted using the Bullet Blender Gold homogenizer (Next Advance, Troy, NY) at a speed of 10 for 5 min at 4 °C. Samples were then lysed completely by additional probe sonication on ice with 30 pulses at power 4 using a sonic dismembranator (Fisher Scientific, Pittsburgh, PA). The SILAC-hFXN-M internal standard solution (20 μL of 2 μg/mL, 40 ng) was then added to each sample and appropriate amounts of hFXN-M standards 4 ng to 200 ng (4, 10, 15, 20, 30, 40, 80, 100, 150, 200 ng) in 5% BSA solution for preparation of the standard curve^{13 (link)}. Supernatants were removed from the DMP cross-linked beads and tissue and BSA standard samples added into the Sarstedt 2.0 mL LB microtubes containing the DMP cross-linked beads. Samples were then incubated with the beads at 4 °C overnight on the rotator.

A Dynabead suspension (5 mg, 150 μL) was transferred to a Sarstedt 2.0 mL low bind (LB) microtube (Sarstedt, AG Numbrecht, Germany). and washed three times using 500 μL of bead washing buffer (PBS with 0.02% Tween-20). Mouse anti-FXN mAb Ab113691 (40 mg, 80 mL) was diluted using PBS to a final volume of 500 μL in a Sarstedt 2.0 mL LB microtube. The beads were incubated with the mouse mAb at 4 °C overnight on a Mini LabRoller rotator (Labnet International, Edison, NJ). The beads were swirled so that they were and thoroughly suspended, the mAb solution removed, and the beads washed twice with 1 mL of the 0.2 M TEA cross-linking buffer. A solution of DMP was prepared by dissolving 13 mg of DMP in 2 mL cross-linking buffer (freshly made every time). The beads were incubated with the 2 mL DMP solution at room temperature (RT) in a Sarstedt 2.0 mL LB microtube for on the rotator. After 1 h, the DMP solution was removed and the beads washed with 1 mL of quenching buffer (0.1 M ethanolamine, pH 8.0; 301 μL in 50 mL water). This was followed by incubation of the beads with 1 mL of quenching buffer at RT on the rotator for 1 h, removal of the quenching buffer and wishing the beads twice with 1 mL of bead washing buffer. DMP cross-linked beads were used immediately.