Vivaspin centrifugal concentrator columns

MMP expression was analyzed by immunoblot in conditioned medium of LX‐2 cells, generated by cultivating the cells in serum‐deprived medium for 48 h, followed by protein concentration using vivaspin centrifugal concentrator columns (MWCO 3,0 kD, Sartorius GmbH). Protein contents were analyzed according to Bradford using Rotiquant (Roth, Karlsruhe, Germany). Either 20 or 40 μg of protein was loaded on 10% polyacrylamide gels and subsequently blotted on PVDF membranes. For the detection of specific proteins, the following antibodies were used: anti‐MMP‐2 (AF902, 2 μg·mL⁻¹; R&D Systems); anti‐MMP‐9 (MAB‐911, 4 μg·mL⁻¹, R&D); MMP‐7 (IMG‐3873; 0.5 μg·mL⁻¹, IMGENEX); MT1‐MMP/MMP‐14 (D1E4, 1 : 1000, Epitomics); TIMP‐2 (MAB971, 1 : 500, R&D). Protein contents were visualized on a ChemiDoc MP system using the imagelab software for quantification (both from Bio‐Rad Laboratories GmbH, Munich, Germany). For the demonstration of equal loading, the membranes were subsequently stained with Ponceau S (Sigma‐Aldrich). MMP‐2 and ‐9 activity was determined using gelatine containing zymography gels (Thermo Fisher Scientific, Karlsbad, CA, USA) as previously described [41 (link)].