Fig. 3

Identification of FKBP12 in epididymal fluid by FK506 affinity chromatography. Epididymal fluid isolated from the epididymis was incubated with an FK506 matrix in the presence or absence of 100 µM soluble FK506. The washed matrix was resuspended in PBS and protein load buffer, boiled, and electrophoresed on 18% Trisglycine gels. A silver stain of the gel demonstrated prominent binding of a 12 kD protein to the FK506 matrix (left panel). When the epididymal fluid was preincubated with soluble FK506, binding of the 12 kD protein to the matrix was completely abolished. Anti-FKBP12 Western analysis of a duplicate experiment confirmed that the 12 kD protein was FKBP12 (right panel).