Histrap high performance 1 ml columns

Constructs containing the cDNA sequences of all FHR proteins, including both known splice variants of CFHR4, were ordered at Invitrogen (Paisley, UK). Constructs were ordered with an in-frame C-terminal tag coding for six histidine residues (6xHis) and cloned into pcDNA 3.1 vectors (Invitrogen). Proteins were transiently expressed in HEK293F cells with 293Fectin and OptiMEM (Invitrogen), using the Freestyle HEK293F expression system (Invitrogen) according to the manufacturer’s instructions. Five days after transfection, supernatants were collected and recombinant human (rh) FHR proteins were purified by Ni²⁺ affinity chromatography with the use of HisTrap High Performance 1 ml columns (GE Healthcare Life Sciences, Freiburg, Germany) according to the manufacturer’s instructions. Subsequent filtrations using Amicon Ultra Centrifugal Filter Devices (Merck Millipore, Darmstadt, Germany) were performed to obtain highly pure rhFHR-3. Throughout the purification process, purity of the rhFHR proteins was assessed by SDS-PAGE under non-reducing conditions and visualized by silver staining using the SilverQuest Staining kit (Invitrogen) according to the manufacturer’s instructions.

Recombinant human FHR (rFHR) proteins, containing a C-terminal 6×-histidine (6×His) tag, were produced and purified as previously described (26 (link)). For FHR-1, the CFHR1*A allotype sequence was used (33 (link)). In short, proteins were expressed by transient transfection of pcDNA3.1 expression vectors in HEK293F cells, after which proteins were purified from the supernatant by Ni²⁺ affinity chromatography using HisTrap™ High Performance 1 mL columns (GE Healthcare Life Sciences, Freiburg, Germany). rFHRs were filtrated and concentrated using Amicon^® Ultra Centrifugal Filter Devices (Merck Millipore, Darmstadt, Germany).

Rat anti-mouse kappa (RM-19) mAb was from Sanquin Reagents (Sanquin, Amsterdam, the Netherlands). High-performance ELISA buffer (HPE) was provided by Sanquin. Proteins were biotinylated according to the manufacturer’s instructions using EZ-Link Sulfo-NHS-LC-Biotin, No-Weigh Format (Thermo Scientific, Waltham, MA, USA), when indicated. Polyclonal anti-FHR-3 antibodies and mAb clone anti-FHR-3.3 were obtained and characterized as part of a previous study (6 (link)). Recombinant human (rh) FHR proteins, containing a C-terminal 6×-histidine tag, were produced and purified as previously described (6 (link)). In short, proteins were expressed by transient transfection of pcDNA3.1 expression vectors in HEK293F cells, after which proteins were purified from the supernatant by Ni²⁺ affinity chromatography using HisTrap™ High Performance 1 mL columns (GE Healthcare Life Sciences, Freiburg, Germany). rhFHRs were filtered and concentrated using Amicon^® Ultra Centrifugal Filter Devices (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions using appropriate molecular weight cut-offs. For rhFHR-4A, a 100 kDa cut-off Amicon^® Filter was used to further purify rhFHR-4A from any high molecular weight aggregates observed after Ni²⁺ affinity chromatography purification (Figures S1A,B in Supplemental Material).