Samples were denatured by the addition of an equal volume of 0.05 M Tris buffer, pH 6.8, containing 2% SDS, 10% glycerol, 2 mM EDTA, 0.005% bromophenol blue, 0.1 M 4,4’diaminodiphenyl sulfone, 12 M urea solution, at 100°C for 5 min. The samples were subsequently electro-phoresed on 12.5% SDS-polyacrylamide gels, and the separated proteins were electroblotted onto nitrocellulose-coated nylon membrane (Hybond-C; Amersham, Castle Hill, NSW, Australia) at 175 mA overnight (transfer buffer: 20% methanol, 20 mM Tris, 150 mM glycine). Buy Advair Diskus Online
A lane containing molecular weight markers (cat. #17-0446-01; Pharmacia Biotech, North Ryde, NSW, Australia) was removed and stained with Napthol blue black, and the remaining lanes were developed in a manner adapted from Towbin et al. as reported previously. Nonspecific binding sites were blocked by incubating the membrane (buffer A: 10 mM Tris pH 7.4, 150 mM NaCl, 5% BSA, 0.2% Nonidet P-40) for 1 h at 37°C in a shaking incubator. Each membrane was subsequently incubated with one of the antibodies directed against a type IV collagen a chain diluted 1: 2500 in buffer A for 2.5 h at room temperature. Each blot was then washed (three times, 15 min each) in buffer A without BSA, but with the addition of 0.25% deoxycholic acid and 0.1% SDS, followed by a further rinse (once, 10 min) in a solution of 10 mM Tris and 0.15 M NaCl. The appropriate secondary antisera (goat anti-rat IgG; cat. #R5005; Sigma) and goat anti-mouse IgG (cat. #M8645; Sigma) were iodinated using the lactoperoxidase method as described previously and were diluted to 1 X 106 cpm/ml in buffer A. Blots were incubated with the secondary antisera for 45 min at room temperature and then washed as described above and air dried. Blots were subjected to autoradiography (Kodak XAR film) or analyzed using a Molecular Dynamics (Sunnyvale, CA) Phospho-Imager and ImageQuant (Molecular Dynamics) software, version 4.1.