Identification of a 12-kD protein on gels, on autoradiograms, and on blots. Proteins present in Sephacryl S-300 column fractions no. 40 through 51 (concentrated 6-fold) were separated on 3% to 17% gradient nondenaturing (Nonidet P-40) polyacrylamide gels and slowly migrating components (400 to 600 kD) were eluted and then applied to 3% to 17% gradient SDS polyacrylamide gels that were subsequently silver stained. The signal at 12 kD is shown (lane 1). The slowly migrating components (400 to 600 kD) were eluted and used for immunoprecipitation with agarose-linked antiphosphotyrosine (PT-66) and the resulting immunoprecipitate was eluted onto 3% to 17% gradient SDS polyacrylamide gels that were subsequently silver stained. Signals in the lower region of the gel are shown (lane 2). hrFKBP12 was applied to a 3% to 17% gradient SDS polyacrylamide gel that was subsequently silver stained (lane 3). Immunoprecipitated immobilized proteins were allowed to undergo phosphorylation by endogenous protein kinase(s) in the presence of 1 μmol/L [γ-³²P]ATP (4 × 10⁷ dpm) and 20 mmol/L magnesium acetate. After the separation of labeled proteins on 3% to 17% gradient SDS polyacrylamide gels, autoradiograms were prepared. The lower region of the gel is shown (lane 4). Immobilized immunoprecipitated proteins were eluted from the solid support onto 3% to 17% SDS polyacrylamide gels. After development, proteins were transferred to a membrane and these blots were then overlaid with rabbit polyclonal anti-FKBP12 (1 to 10,000). Goat antirabbit antibody conjugated to horseradish peroxidase (1 to 70,000) was added and detection was by enhanced chemiluminescence (ECL). The lower region of the blot is shown (lane 5). hrFKBP12 was applied to a 3% to 17% gradient SDS polyacrylamide gel and transferred to a membrane, and the blot was overlaid with anti-FKBP12 and detected as above (lane 6). The 6.5-kD molecular mass markers are shown.