Affinity Purification and FKBP1A Ligand Binding Assay

CHO-DSP1 cells were transfected with control plasmid pcDNA 3.1 (+) or plasmid pcDNA3.1_huFKBP1A-His-Myc [83 (link)]. At 24 hrs post-transfection, cells were lysed in lysis buffer [50 mM NaH₂PO₄ (Fisher Scientific), 300 mM NaCl (Fisher Scientific), 10 mM imidazole (Sigma Aldrich), 0.05% Tween20 (Sigma Aldrich), pH 8.0] with cOmplete EDTA-free protease inhibitor (Sigma Aldrich) for 30 min on ice. The sample was centrifuged at 3,000 RCF for 10 min at 4°C to remove cell debris, and the supernatant was saved as cell extract. Ten volumes of cell extract were precipitated with 1 volume of pre-equilibrated Ni-NTA agarose beads (Qiagen) at 4°C overnight, followed by washes using wash buffer [50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, 0.05% Tween20, pH 8.0]. Meanwhile, MeWo-DSP1 cells were extracted using the above lysis buffer and treated with DMSO, 10 μM of tacrolimus, 10 μM pimecrolimus or 10 μM sirolimus for 30 min at 4°C with end-over-end rotation, followed by incubation with the above-prepared FKBP1A-retaining Ni-NTA agarose beads at 4°C overnight. The bound proteins were eluted from the beads using elution buffer [50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, 0.05% Tween20, pH 8.0], followed by western blotting analysis.