Amicon ultra 15ml centrifugal devices

The sFlt-1 sequence for the first 3 lg-like extracellular domains of sFlt-1 (3) [15 (link)] was cloned into the pFastBac1 plasmid (Life Technologies) and then transformed into DH10Bac E.coli, which were plated on triple antibiotic plates containing kanamycin (50 μg/mL), gentamicin (7 μg/mL, Sigma Aldrich), tetracycline (10 μg/mL, Sigma Aldrich), IPTG (40 μg/mL, Sigma Aldrich) and Bluo-gal (100 μg/mL, Thermo Fisher Scientific). The sFlt-1 (3) gene-containing bacmid was isolated from DH10Bac E.coli (Life Technologies) and transfected into SF9 insect cells for virus production (provided by the Tissue Culture Facility, UC Berkeley). Virus was then used to infect High Five insect cells (provided by the Tissue Culture Facility, UC Berkeley) to induce sFlt-1 (3) protein expression. After 3 days, protein was purified from the supernatant using Ni-NTA agarose beads (Qiagen Laboratories). Recombinant sFlt-1(3) was eluted from the Ni-NTA beads using an imidazole gradient and then concentrated and buffer exchanged with 10% glycerol/PBS using Amicon Ultra-15mL Centrifugal devices (EMD Millipore). The protein solution was sterile filtered and the concentration was determined using a BCA assay (Thermo Fisher Scientific). The molecular weight of sFlt-1(3) was determined from a 4-20% gradient SDS-PAGE gel. For convenience, we refer to this protein as sFlt in this manuscript.