Figure 1From: Fluorometric immunocapture assay for the specific measurement of matrix metalloproteinase-9 activity in biological samples: application to brain and plasma from rats with ischemic strokeFRET peptide-based immunocapture assay for MMP-9. Mouse monoclonal anti-MMP-9 was added to a 96-well plate coated in Protein G to facilitate binding to the Fc region of the antibody, thus leaving the Fab region in an optimal orientation for tightly binding MMP-9. After incubation with the antibody, biological samples containing both active and pro-MMP-9 were added to the plate and allowed to incubate with the antibody to facilitate tight binding. In experiments that required activation of pro-MMP-9 to quantify total levels, APMA is added to activate the pro-MMP-9. Intact FRET peptide, in which the QXLâ„¢520 fragment is quenching the fluorescence of the 5-FAM donor, is added to the wells. Catalytically active MMP-9 is able to cleave the FRET peptide, allowing the fluorescent 5-FAM fragment to be read and monitored at excitation/emission wavelengths of 485/528Â nm.Back to article page