Quantifying TGFβ Ligand from Bone Lysates

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.

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Schurman C.A., Kaya S., Dole N., Luna N.M., Castillo N., Potter R., Rose J.P., Bons J., King C.D., Burton J.B., Schilling B., Melov S., Tang S., Schaible E, & Alliston T. (2024). Aging impairs the osteocytic regulation of collagen integrity and bone quality. Bone Research, 12, 13.

Publication 2024

Bead Mill Tubes Pierce Coomassie (Bradford) Assay Kit Bio-Plex Pro TGF-β Assay Kit Bio-Plex 200

Corresponding Organization :

Other organizations : University of California, San Francisco, Washington University in St. Louis, Buck Institute for Research on Aging, Lawrence Berkeley National Laboratory, University of California, Berkeley

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Variable analysis

dependent variables

Total levels of TGFβ ligand in hydrolyzed lysates

control variables

Protein harvesting method using Bead Mill Tubes with RIPA buffer and protease inhibitor
Homogenization using a benchtop bead mill homogenizer at 4 °C
Protein concentration quantification using the Pierce Coomassie (Bradford) Assay Kit

controls

Positive control: None specified
Negative control: None specified

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