Clone lh7

Isolated EOM was solubilized with SDS PAGE sample buffer and subjected to Western blotting on 7.5% or 4–20% gradient gels (8 (link)). After transfer to PVDF membranes, the blots were developed with rabbit polyspecific antibodies against the α2 chain of type IV collagen (T-15, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or the helical domain of α1(IV) and α2(IV) chains (M3F7, 1:500, Developmental Studies Hybridoma Bank, University of Iowa, IA). Type VII collagen antibodies used were from Millipore (Billerica, MA) (1:500, #234192) and from Sigma-Aldrich (St. Louis, MO) (1:500, clone LH7.2). For comparison, whole crowns (minus roots) were pulverized and extracted with 4 M guanidine HCl and 0.5 M EDTA containing protease inhibitors (9 (link)); the lyophilized extract was solubilized with SDS PAGE sample buffer and subjected to Western blotting with rabbit anti-MMP-20 antibodies specific to the N-terminal domain (1:2000, Sigma-Aldrich, #M-5934) and C-terminal domain (1:200, Origene, Rockville, MD, clone EP1275Y), and to casein gel zymography (10 (link)). Casein gels were visualized after staining with Coomassie brilliant blue dye.

Pepsin digested and control protein extracts from whole crowns and enamel matrixes were dissociated in SDS/8M urea sample buffer, heated at 95°C, and electrophoresed as previously described using 4–20% linear gradient and 7.5% gels [26 (link)]. Blots were incubated with either primary mouse anti-type VII collagen monoclonal antibody recognizing the non-helical carboxy terminal region of the collagen VII dimer (1:500, clone LH7.2, Sigma-Aldrich) or primary rabbit anti-type VII polyclonal antibody (1:500, catalog #234192, Millipore), and then subsequently with horseradish peroxidase-conjugated goat-anti-mouse or goat-anti-rabbit IgG (1:10,000, BioRad, Irvine, CA) secondary antibodies, respectively. Chemiluminescent digital images were captured with a Fuji LAS-4000 imager (GE, Piscataway, NJ).