Bradford dye binding procedure

Cells for isolation of total proteins were harvested at the same time-points and from the same cultures as that used for RNA preparation (Northern blot analysis described above). One milliliter aliquots were harvested and kept at −80 °C, until all samples were collected. The cell pellets were thawed on ice, suspended in 50 mM TrisHCl, pH = 7.4, to a calculated OD₆₀₀ of 10.0. PMSF, Dnase, Rnase, and lysostaphin were added to the samples, and they were incubated at room temperature for 15 min. Cellular debris was removed by centrifugation and the protein concentration of each sample was measured using the Bradford dye-binding procedure from Bio-Rad. For immunoblotting, a total of 5 μg of each sample was loaded on NuPAGE^® 4–12% Bis-Tris gels (Invitrogen), using MOPS-Buffer (Invitrogen). The proteins were blotted onto a polyvinylidene difluoride membrane, using the XCell II blotting module (Invitrogen). The membranes were pre-blocked with IgG, before probing with antibodies directed against the Rot transcriptional regulator (Rabbit-anti-Rot-antibody [37 (link)]) at a 1:2000 dilution. Bound antibody was detected with the WesternBreeze Chemiluminescent Anti-Rabbit kit or Anti-mouse kit (Invitrogen). The Western blot analysis was repeated three times with similar results.

For Western blots, testis and seminoma samples (standardized ratio: 100 mg wet weight/400 μm buffer containing 1% SDS and 4% 2-mercaptoethanol) were extracted as previously described in detail by Bräuer et al. [12 (link)]. The protein content of these samples, and of the collected seminal fluid samples, was measured with a protein assay based on the Bradford dye-binding procedure (BioRad, Hercules, CA). Total protein (100 μg) was then analyzed by Western blot. Proteins were resolved by reducing 15% SDS-polyacrylamide gel electrophoresis, electrophoretically transferred at room temperature for 1 h at 0.8 mA/cm² onto 0.1 μm pore size nitrocellulose membranes and fixed with 0.2% glutaraldehyde in phosphate-buffered saline for 30 min. Bands were detected with primary antibodies to SP-A (1:500), SP-B (1:500), SP-C (1:500), SP-D (1:500, 0,5μg/μl) and secondary antibodies (anti-rabbit/anti-mouse IgG, respectively, conjugated to horseradish peroxidase, 1:5000) using chemiluminescence (ECL-Plus; Amersham-Pharmacia, Uppsala, Sweden). Human lung was used as the control. The molecular weights of the detected protein bands were estimated using standard proteins (Prestained Protein Ladder, Fermentas, St. Leon-Rot, Germany) ranging from 10 to 170 kDa.