Protein a conjugated sepharose

Fcp5 protein was produced by transient transfection of HEK293T/17 cells (ATCC, Manassas, VA, USA) in 100-mm tissue culture dishes using 6 µg of plasmid DNA with 20 µg of linear polyethylenimine (Polysciences, Warrington, PA, USA) per dish and culturing 9 days in DMEM/F12 (Lonza, Walkersville, MD, USA) with 2% (v/v) immunoglobulin-depleted fetal bovine serum (Thermo-Hyclone, Logan, UT, USA) and penicillin–streptomycin (Lonza), with media changes every 3 days. The collected cell supernatants were clarified by centrifugation at 1,500 × g for 10 min and the secreted Fcp5 purified by affinity chromatography using protein A-conjugated Sepharose (GE Healthcare, Pittsburg, PA, USA) with elution by 0.1 M glycine, pH 3.0, followed by dialysis in PBS and quantitation by Coomassie blue protein assay (Thermo-Pierce, Dallas, TX, USA). The integrity of the purified Fcp5 was documented by SDS-PAGE on native and reduced samples with staining by Coomassie brilliant blue, or by periodic acid Schiff staining for carbohydrate (Thermo-Pierce).

Cells were lysed in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 100 mM HEPES, 5 mM Na₄P₂O₇, 5 mM NaF, 2 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 10 mg/l aprotinin, and 10 mg/l leupeptin), and cleared cell lysates were incubated with Protein G-conjugated Sepharose or Protein A-conjugated Sepharose (GE Healthcare, Piscataway, NJ, USA) and the appropriate antibody overnight at 4 °C. Following incubation, the resin was washed three times with co-immunoprecipitation lysis buffer, and protein samples were eluted by boiling in 5 X SDS sample buffer for western blotting analysis.