2.7. 4-Hydroxynonenal (HNE)-Protein Conjugate Assay

The accumulation of 4-HNE-protein conjugates represents the local oxidative status and lipoperoxidation process. At high concentration, 4-HNE is a bioactive toxic end-product of lipoperoxides that is reported as important pathomechanistic factor in many disease [12, 13]. As to its exceptional reactivity, its conjugates with proteins can be assessed by specific antibodies against the conjugate in western blot. Proteins were extracted from 30 μl of vitreous and 30 μl aqueous humor samples in presence of a protease inhibitor cocktail (Complete®, Roche Diagnostics S.p.A., Milano, Italy) in order to inhibit a broad spectrum of serine-, cysteine-, and metalloprotease activities, centrifugated at 2000 ×g at 4°C for 10 min to eliminate insoluble residues, and quantified for protein content by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). As previously described [8], protein was extracted with SDS-containing modified Laemmli buffer (final concentrations of Tris-HCl 60 mM, EDTA 1 mM, 5% glycerol, and SDS 2%), pH 6.8, at 95°C for 5 min. Aliquots were kept at −20 C until use and β-mercaptoethanol (5% v/v, final concentration) was added before SDS-PAGE. The extracted proteins were requantified, loaded at 20 μg/lane, and separated in a 10% polyacrylamide/acrylamide (w/v) SDS-PAGE. Separated proteins were transferred onto a nitrocellulose membrane (Amersham Biosciences, Fairfield, Connecticut), and equality of loaded and transferred protein amounts and of protein pattern of all sample lanes was verified by Ponceau S staining. Image acquisition, protein band intensity quantification, and comparison of lanes were performed using ImageJ software (version 1.46, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Membranes were blocked with bovine serum albumin (BSA; dissolved at 5% (w/v) in phosphate-buffered saline containing 0.1% (v/v) Tween) for 1 h and subjected to the mouse monoclonal anti-4-HNE-conjugate antibody at 1 : 2000 dilution (clone HNEJ-2, Abcam, Cambridge, UK) overnight at 4 C [11, 14, 15]. Then membranes were washed with PBS-Tween 0.1% and incubated with the secondary anti-mouse horseradish peroxidase-conjugated antibody (Amersham, Bucks, United Kingdom) at 1 : 20000 dilution for 1 h at room temperature. The PBS-Tween 0.1%-washed membranes were analyzed for antibody-positive bands which were visualized by enhanced chemiluminescence (ECL) and acquired and quantified with Chemidoc MP equipment (Bio-Rad Laboratories, Hercules, California), using the PDQuest software (Bio-Rad, version 7.2) according to manufacturer's instructions. 4-HNE-modified human serum albumin (4-HNE-HSA) [14, 15] was prepared as reference sample for normalization of sample conjugate values. For this, 0.1 μg 4-HNE-HSA was run in parallel with samples in each gel. For densitometry analysis, the values obtained for all 4-HNE-positive bands of a sample were referred to the reference 4-HNE-HSA band, summarized in each lane, and expressed as 4-HNE arbitrary units for each patient.