Quantifying Protein Expression in Mineralized Scaffolds

Three of each cell-seeded mineralized collagen and mineralized collagen–amnion scaffold were lysed for protein at Days 0, 4, 7, 14 and 28. To extract protein, a solution of phosphate inhibitor cocktails (Sigma-Aldrich) and an RIPA lysis buffer were used [42 (link)]. To evaluate the protein concentration, a Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific) and a Pierce^TM Bovine Serum Albumin Standard Pre-Diluted Set (Thermo Fisher Scientific) were used. Analysis of protein activity was quantified with a Western Blot assay, loading 5 µg of protein lysate in each lane and using a SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) or a SuperSignal West Pico PLUS Sensitivity Substrate (Thermo Fisher Scientific) to visualize bands. Images were captured with an Image Quant LAS 4010 machine (GE Healthcare Life Sciences, Little Chalfont, UK). Primary and secondary antibodies are listed in Table 1, and β-actin was used as a loading control.