Anti peroxiredoxin 2

In each case three micrograms of vitreous sample protein was separated on Novex 10–20% gradient Tris-Glycine polyacrylamide gels (Invitrogen Corporation, Carlsbad, CA) and subsequently transferred to nitrocellulose Hybond-C Extra membranes (GE Healthcare). The membranes were blocked overnight with 5% skimmed milk in 80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl, and 0.05% Tween 20 buffer, pH 7.5. Membranes were incubated with anti-albumin (Genway Biotech, CA, USA; 1 : 5000) and anti-peroxiredoxin 2 (Abcam, Cambridge, UK; 1 : 200). No suitable antibodies were commercially available for the rabbit F isoform of α-1-antiproteinase or the rabbit collagen-Iα1 fragment that was identified with LC-MS/MS. Following washing, the membranes were further incubated with appropriate horseradish peroxidase-conjugated secondary antibodies: P0163 sheep and P0260 mouse (both 1 : 1000; DAKO, Glostrup, Denmark). Proteins were visualized with the enhanced chemiluminescence system (GE Healthcare) and imaging system (Fujifilm LAS-3000, Tokyo, Japan).