Extraction of basement membrane, bacterial collagenase solubilization and the analysis of native type IV collagen NC1 domains from different species. (A) Native protein (60 μg per lane) from the 6 different species and 250 ng of recombinant human α3(IV)NC1, (rh-α3(IV)NC1)³⁵  was run on a 15% SDS-PAGE under extreme reducing (10% β-mercaptoethanol) conditions, and incubated with the anti-36mer antibody.²³  Monomer forms (M) of NC1s are detected in all lanes. Although subjected to extreme reduction, significant amounts of the isolated NC1 remain as dimers (D). (B) C elegans NC1 in dimeric form (D) was visualized after increased film exposure. (C) The same samples were run on a 15% SDS-PAGE under nonreducing conditions and incubated with 3 different Goodpasture sera (1:20 dilution). A representative blot is shown. As expected, sera from Goodpasture patients bound recombinant human α3(IV)NC1 (lane 1). Mouse, chicken, and frog kidney BM extracts show strong reactivity with Goodpasture sera (lanes 2-4). NC1 in monomer form (M) could only be detected for chicken without the use of a reducing agent, whereas for the other species the Goodpasture sera detected dimers (D) under nonreducing conditions. Interestingly, no reactivity to Danio rerio, Drosophila, and C elegans NC1 was found with any of the analyzed Goodpasture sera (lanes 5-7). (D) Reduction of the samples eliminates the conformational structure of the Goodpasture epitope. With slight reduction (1.5% β-mercaptoethanol) some monomer (M) from each of the 3 positive species (Mus, Gallus, Xenopus) native NC1s was recognized by the Goodpasture sera. (E) Under extreme reducing conditions (10% β-mercaptoethanol), binding to all monomer and dimer forms of NC1 by Goodpasture sera was eliminated, as expected and as previously described.¹⁷