Quantification of MMP-13 and ANKH Proteins

The protein levels of MMP-13 and ANKH were determined by Western blot. Total protein was extracted by homogenization with the complete Lysis-M kit (No. 04719956001; Roche) from bovine menisci and quantified by the BAC Protein Assay Kit (Pierce). Equal amounts of protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes for immunoblot analysis and stained with specific primary antibodies. The following primary antibodies were used: MMP-13 antibody (H-230, Lot D1113; Santa Cruz Biotechnology) and ANKH antibody (ab90104; Abcam). Fluorescence-labeled secondary antibodies were detected with a fluorescence scanner (Odyssey; LI-COR Biosciences). Band densities were quantified using Image Acquisition and Analysis Software (UVP). Parallel gels were prepared for Coomassie Blue staining to confirm equal loading of the samples as described.^{43 (link)} Equal amounts of protein were electrophoresed in 10% SDS-PAGE, and the gel was prefixed in 50% MeOH, 10% HoAC, and 40% H₂O for 30 minutes and then stained with 0.25% Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories) in the above solution for 4 hours. The gel was destained in 5% MeOH, 7.5% HoAC, and 87.5% H₂O until the background was clear. The destained gel was stored in 7% HoAC, and a photograph was taken using a Canon camera (SD-1000; Canon Inc).