Recombinant mmp 2

Aneurysm samples were homogenized in protein extraction buffer (0.3 M sucrose; 25 mM Imidazole, 1 mM EDTA, pH 7.2 complete protease inhibitor cocktail 2 and 3, Sigma Aldrich, Søborg, Denmark). Samples were centrifuged for 10 min at 6000× g at 4 °C. Protein concentration was determined by Bicinchoninic Acid Kit for Protein Determination (Sigma Aldrich, Søborg, Denmark) using bovine serum albumin as the standard. A total of 12 µg protein samples and 1.25 µL recombinant MMP-2 (Sigma Aldrich, Søborg, Denmark) were mixed with an equal amount of 2× tris-glycine SDS sample buffer (Thermo Fischer) loaded onto a Novex zymogram gel containing 10% gelatin (Thermo Fisher, Slangerup, Denmark) and proteins were separated by gel electrophoresis at 125 V for 90 min. Proteins were then allowed to refold 30 min in renaturation buffer (Thermo Fisher, Slangerup, Denmark) followed by 24 h at 37 °C in developing buffer (Thermo Fischer, Slangerup, Denmark). Finally, undigested proteins in the gel were stained with simple blue stain (Thermo Fisher) for 30 min. White bands were inverted and quantified in Molecular Imager Image Lab (ChemiDoc WRS+, Biorad, Copenhagen, Denmark).

Sodium alginate (ALG, low viscosity, 4-12 cP), sodium periodate (NaIO₄), dopamine hydrochloride (DOPA), N-phenyl-p-phenylenediamine, and recombinant MMP-2 were purchased from Sigma-Aldrich. Thiol-modified hyaluronic acid (HA-SH, Mw = 300 kDa, degree of thiol substitution = 33%) was purchased from ESI BIO, USA. MMP-SP (Seq: CGPLGGGRMSMPVRDGSC where C is a thiol-containing cysteine and GGRMSMPV is the MMP-cleavable sequence.) was purchased from Bankpeptide Ltd., Hefei, China. DPCA was purchased from Santa Cruz Biotechnology Ltd. L929 Mouse fibroblast cell line (L929) and rat cardioblasts (H9C2) were purchased from National Biomedical Experimental Cell Bank, Beijing, China.

Proteins were extracted by homogenization in an extraction buffer. The bicinchoninic acid protein determination kit (Sigma-Aldrich, Søborg, Denmark) was used to determine protein concentrations using serum albumin as standard. AAA protein samples (12 µg) and 1.25 µL recombinant MMP2 (Sigma Aldrich, Søborg, Denmark) were loaded onto Novex zymogram gel, separated, renatured, and incubated for 24 h at 37 °C in a developing buffer, before undigested proteins were stained by a simple blue stain (Thermo Fischer, Waltham, MA, USA). White bands representing digested proteins were inverted and quantified in Molecular Imager Image Lab (ChemiDoc WRS+, Biorad, Copenhagen, Denmark) as described by Melin et al. [30 (link)].