Amicon ultra 2 centrifugal unit

Before performing ProtoArray microarrays, recombinant (r) PCSK9 (carrier-free; R&D Systems; Minneapolis, MN) was biotinylated using the EZ Link Sulfo-NHS-SS Biotinylation kit (Pierce Thermo Scientific; Rockford, IL). First, to exchange buffers, 10 μg of rPCSK9 was added to a Zeba™ spin desalting column that has been pre-equilibrated according to the manufacturer’s protocol. Centrifugation was performed at 1000 × g for 2 minutes, and the resulting flow-through that contained the rPCSK9 protein in PBS was used in the next step. Sulfo-NHS-SS-Biotin was prepared by dissolving in ultrapure water at the concentration of 10 mM. Biotinylation of rPCSK9 was done with an at least 40-fold molar excess of Sulfo-NHS-SS-Biotin for 1 hour at room temperature. Filtering through another pre-equilibrated Zeba™ spin desalting column was performed as described above to remove excess biotin. Alternatively, excess biotin was removed by filtering through a 3 kDa Amicon ultra-2 centrifugal unit (Fisher Scientific; Pittsburg, PA). Protein concentrations were measured using a NanoDrop 2000 (Fisher Scientific). A HABA assay (reagents provided with the Biotinylation kit) was used to measure biotinylation levels. Calculations for the HABA assay were performed according to the protocol provided with the assay.