Analysis of Protein Complexes
Recombinant human FS, rh-inhibin A, and rh-activin A standards, tissue homogenates, and urine samples were subjected to SDS-PAGE in a 4-20% or 12% Tris-glycine gel. Tissues were homogenized in a Tris buffer containing 1 mM EDTA, 10 ^g/ml pepstatin A, 0.25 M sucrose, and 17.4 ^g/ml PMSF, and total protein amounts were equivalent per gel lane loaded. Samples were reduced in 10% SDS sample buffer with 2.5% (3-mercaptoethanol and then heated at 90°C for 2 min. The gels were fixed in a 10% acetic acid/40% ethanol solution before staining with silver nitrate reagents (serially in 1.7 mM sodium dithionite for sensitization, 12 mM silver nitrate/0.025% formaldehyde solution, then 40 mM sodium thiosulfate pentahydrate solution for development) or were transferred to polyvinylidene fluoride membranes for immunoblot analysis. flovent inhaler
The monoclonal antibodies used to detect FS via immunoblot (Western) analysis were 7FS30 and 6FS6, either alone or in combination, and immunoreactivity was determined using ECL (enhanced chemiluminescence) detection (Amersham, Arlington Heights, IL). The primary antibodies used in the experiment were biotinylated, and incubation of the blot with horseradish peroxidase (HRP)-streptavidin by itself did not result in the detection of any protein bands. A secondary rabbit anti-mouse antibody conjugated with HRP was used in the pituitary and ovarian studies. Cross-linking of proteins was done as previously described.