Figure 3. Purification of nCFH from the retina of a deceased individual with AMD (83 year old, female, AREDS Grade 1).

a. Lane 1 demonstrates a Western blot of whole protein extract from the AMD retina probed with the C166 antibody. Lane 2 affinity purified sample obtained by applying the whole protein extract from the AMD retina to an anti-nCFH monoclonal antibody affinity column probed with the C166 antibody. Lane 3, the affinity purified sample probed with a specific antibody to CFH. b. Silver stain of the affinity purified AMD retinal extract. The two arrows indicate the bands that were excised and subjected to tandem mass spectrometry to identify the nitrated residues. c. Western blot of whole protein extract from distinct regions of the retina from a patient with AMD (93 year old, female, AREDS Grade 4) probed with the C166 monoclonal antibody.1 = retina without macula, 2 = macula retina (8 mm trephine), 3 = choroid/RPE from below macula (8 mm trephine), 4 = choroid/RPE from rest of eye. d. An example tandem mass spectrum showing the detection of NO₂Y1177 (Domain 20), from the affinity purified sample (b).