Category Archives: Biological Fluids - Part 2

Identification of Naturally Occurring Follistatin Complexes: MATERIALS AND METHODS(4)

The inhibin B standard was prepared by immunoaf-finity purification from human follicular fluid and was provided by Dr. Nigel Groome. No cross-reactivity between heterologous ligands was found for any of the assays using purified recombinant proteins. Recombinant human ligands were added at two dilutions into human urine from a pool of men. Inhibin A recovery was 100% and 110% for high and low standards. Inhibin … Continue reading

Identification of Naturally Occurring Follistatin Complexes: MATERIALS AND METHODS(3)

Immunoassays Unbound FS was measured in 96-well plates that were coated with 4 ^g/ml of the monoclonal antibody 6FS7. Standards and samples were added coincident with a bio-tinylated secondary antibody, monoclonal 7FS30. The an-tibody-antigen mixture was allowed to incubate overnight on a shaker at room temperature. After an overnight incubation, the plate was washed and incubated with HRP-streptavidin. The enzyme was incubated for 2 h … Continue reading

Identification of Naturally Occurring Follistatin Complexes: MATERIALS AND METHODS(2)

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 … Continue reading

Identification of Naturally Occurring Follistatin Complexes: MATERIALS AND METHODS(1)

Reagents and Sample Collection Recombinant human (rh) activin A and rh-FS288 were provided through the National Pituitary and Sormone Distribution Program (Rockville, MD) of the NIH. Recombinant human inhibin A was generated in the laboratory. Antibodies that recognize FS, activin, and inhibin used in this study have been previously described. Normally cycling and timed-pregnant Sprague-Dawley-derived female rats were obtained from Harlan BioSciences (Indianapolis, IN). The … Continue reading

Identification of Naturally Occurring Follistatin Complexes in Human Biological Fluids

Follistatin (FS) is a monomeric glycoprotein that binds activins and inhibins. Since initial identification of FS as an inhibitor of FSH release, it has become clear that the role of FS action in the pituitary lies in its ability to bind and regulate activins. Activins and inhibins are disulfide-linked dimeric protein members of the transforming growth factor p (TGF-p) superfamily. Inhibins are he-terodimeric proteins composed … Continue reading