Amicon ultracel 100 k

Recovered His::PfPLSCR protein was refolded by rapid dilution into IMAC buffer A [250 mM NaCl, 0.5 mM CaCl₂, 1 mM TCEP, 0.1 % Fos-choline-12 (n-Dodecyl-phosphocholine, Anatrace), 25 mM HEPES pH 7.5], resulting in a final SDS concentration of 0.1 %. Refolded protein was filtered through a 0.45 μm filter prior to immobilised metal affinity chromatography (IMAC) using a HisTRAP™ High Performance column (GE Healthcare), which was equilibrated with IMAC buffer A.
The loaded protein column was washed with several column volumes of wash buffer [IMAC buffer A + 0.4 % SDS] and IMAC buffer A. The protein was eluted in a linear imidazole gradient using IMAC buffer B [IMAC buffer A + 1 M imidazole]. Fractions containing the desired protein were concentrated (Amicon Ultracel 100 K, Millipore) and further purified by size-exclusion chromatography (SEC) using a Superdex S200 10/300 G L column (GE Healthcare) in SEC buffer [250 mM NaCl, 0.5 mM CaCl₂, 1 mM TCEP, 0.1 % FC-12, 25 mM HEPES pH 7.5]. All buffers contained cOmplete™ EDTA-free protease inhibitors (Roche). Purity of the protein was assessed by SDS-PAGE using NuPAGE™ Novex® Bis-Tris gels and blue native PAGE was performed by using the NativePAGE™ Novex® Bis-Tris Gel System and G250 additive (Life Technologies).

Samples were fixed with paraformaldehyde (20% v/v), transferred into Amicon centrifuge filters (Amicon Ultracel 100 k, regenerated cellulose membrane, Millipore, Carrigtwohill, CO, Ireland) and centrifuged for 10 min at 1000×g. The pellets were resuspended and washed twice in PBS (phosphate-buffered saline). The cells were recovered by centrifugation at 3000×g for 15 min. After the addition of PicoGreen^® (Quant-iT PicoGreen^® dsDNA, Life Technologies, Grand Island, NY, USA), the cells were counted in 96-well plates along with 50 μL of Sphero™ AccuCount blank beads (Spherotech, Lake Forest, IL, USA). Cell density was estimated with an LSRII flow cytometer (BD, Sparks, MD, USA) using a 488-nm laser. A calibration curve relating the ratio of cell events to bead events and the cell density was constructed using a serial dilution of a cell sample. Density was then determined by microscopy (Helber Bacteria Cell counting chamber with Thoma ruling, Hawksley, Lancing, Sussex, UK). The OD₆₆₀ of TIE-1 cells vs. cell numbers were plotted to obtain a standard curve.