Pierce coomassie bradford assay kit

60 μM GAPR-1 was incubated with 10 mM NEM at room temperature for 1 h in the dark. Unreacted NEM was removed by three repetitive steps of dilution in NT-50 buffer and concentration using Amicon 10 kDa MWCO centrifugal filter (Merck, Darmstadt, Germany). Protein concentration was determined using Pierce Coomassie (Bradford) assay kit (Thermo Fisher, Eindhoven, the Netherlands) and the degree of NEM-modification was determined by subsequent PEG-maleimide labelling and SDS-PAGE.

Protein from bone lysates (12-15 per age and sex) was harvested as previously described^{86 (link)} using Bead Mill Tubes (Thermo Fisher Scientific, Waltham, MA) containing radioimmunoprecipitation (RIPA) buffer with eComplete Mini protease inhibitor (Roche, Basil Switzerland). and homogenized using a benchtop bead mill homogenizer (Omni International, Kennesaw, GA) at 4 °C. Serum was collected from blood as previously described.^{27 (link)} Protein concentrations were quantified using the Pierce Coomassie (Bradford) Assay Kit (Thermo Fisher Scientific, Waltham, MA). Total levels of TGFβ ligand were quantified in hydrolyzed lysates by using the Bio-Plex Pro TGF-β Assay Kit (Bio-Rad, Hercules, CA) on a Bio-Plex 200 (Bio-Rad, Hercules, CA). TGFβ ligand levels were normalized to each sample’s original total protein concentration.