Talon superflow affinity resin

The fractions containing Trf2 and TFIIA/ALF from the gel filtration were pooled and subjected to tandem Flag-His purification. Tagged Trf2 was immunoprecipitated with anti-Flag M2-agarose (Sigma), eluted with Flag peptide (0.5 mg/mL), and further affinity purified with Talon superflow affinity resin (Clontech) and elution with 0.1 M EDTA. Purified complexes from the Trf2^tag/tag and control Trf2^+/+ fractions were resolved by SDS-PAGE and stained using the Silver Quest kit (Invitrogen). Sections of gel from the lowest to highest molecular mass were systematically excised and analysed by mass-spectrometry either at the Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, MA) or the IGBMC mass-spectrometry facility.

FLAG epitope-tagged recombinant human WT PCSK9 along with A44P, R46L, R496W, and Δ31–52 variants were produced in stably transfected HEK293S cells and purified from conditioned culture medium as described previously (18 (link)). Fluorescently labeled WT PCSK9 was prepared using the DyLight800 antibody-labeling kit (Thermo Fisher Scientific) as per the manufacturer's instructions followed by gel filtration chromatography on a Superdex 200 10/300 GL column (GE Healthcare) to remove unbound dye. The extracellular domain (ECD) of LDLR used for PCSK9 binding assays was partially purified from conditioned medium of HEK293S cells cultured as described (18 (link)) stably transfected with a plasmid encoding amino acids 1–692 of human LDLR containing a C-terminal His₆ tag (a kind gift from R. Milne, University of Ottawa). LDLR-ECD was enriched by affinity chromatography using TALON superflow affinity resin (Clontech) followed by gel filtration chromatography on a Superdex 200 10/300 GL column (GE Healthcare).