Digoxigenin nhs

cDNA encoding a truncated human ACE2 fused to human albumin was sub-cloned into pFUSE2ss-CLIg-hk (InvivoGen). The vector was transiently transfected into Expi293F cells in suspension (Thermo Fisher Scientific) using the ExpiFectamine 293 transfection kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cells were cultured for 7 days at 37 °C with 80% humidity and 8% CO2 on an orbital shaker platform set to 125 rpm before the medium was collected. The secreted fusion protein was purified on a CaptureSelect™ human albumin affinity matrix (Life Technologies), and protein eluted by adding 20 mM Tris and 2.0 M MgCl2, pH 7.0 before up-concentration using Amicon® Ultra-15 50K Centrifugal Filter Units (Merck Millipore). Buffer exchange to PBS was performed before size exclusion chromatography (Äkta Avant, GE Healthcare) with a SuperdexTM 200 Increase 10/300 GL (Cytiva) prior to up-concentration using Amicon® Ultra-0.5 Centrifugal Filter Units (Merck Millipore). The protein eluted as a dimer. For hapten-conjugation, digoxigenin-NHS (Sigma Aldrich, cat. No 11333054001, 40μg/mg protein) was added to protein solublized in PBS. After 30 min of incubation at 22 °C, free digoxigenin was removed using Amicon® Ultra-0.5 Centrifugal Filter Units.

In total, 10 mg streptavidin (Sigma) was dissolved in sodium bicarbonate pH 8.2. 1 mg digoxigenin-NHS (Sigma, 55865) was dissolved in 100 μl dimethyl sulfoxide, mixed into streptavidin solution, and incubated 3 hours at room temperature rotating. Unconjugated digoxenin was removed by exchanging buffer five times with 10 ml PBS each round using a spin concentrator (Vivaspin, 20 ml, 30 kDa MWCO). streptavidin-digoxigenin conjugate was finally concentrated to 3 mg/ml and stored at 4 °C.