Quantikine elisa gdf 8 myostatin immunoassay

Measurement of MIP-2 concentration was performed on gastrocnemius muscle protein extracts. Protein extracts were obtained homogenizing minced muscle tissues in tissue lysis buffer (150 mM Tris-HCl, pH 7.5; 1 mM EDTA, 1% Triton, 150 mM NaCl (all from Sigma-Aldrich)) for 30 s, followed by lysis on ice for 30 min and by centrifugation at 10,000 r.p.m. at 4 °C to pellet cell debris. Supernatant was quantified using DC Protein Assay (Bio-Rad) and 500 μg of proteins were loaded to perform a MIP-2 ELISA assay (R&D Systems), following manufacturer’s instructions. Myostatin levels in serum were measured using Quantikine ELISA GDF-8/Myostatin Immunoassay (R&D Systems), following manufacturer’s instructions. Blood samples were allowed to clot O/N at 4 °C before centrifuging for 20 min at 2000×g. Serum samples were separated and maintained at −20 °C before loading on the ELISA plate.