Histrap hp 5 ml

After protein expression and lysis of the obtained E. coli cells, the cleared lysates could be directly loaded onto an Äkta chromatography system for protein purification (GE Healthcare). The lysis buffer was supplemented with 20 mM imidazole to reduce aspecific binding to the column (HisTrap HP 5 ml, GE Healthcare) for immobilized metal affinity chromatography (IMAC). The column was equilibrated with 20 mM NaH₂PO₄, 0.5 M NaCl and 20 mM imidazole at pH 7.5 before sample loading and then washed after sample loading for 5 column volumes with 8% elution buffer (100% elution buffer contained 20 mM NaH₂PO₄, 20 mM NaCl, 400 mM imidazole at pH 7.5). Sample elution was done using 100% of the elution buffer and the obtained fractions were subjected to SDS-PAGE followed by WB. Fractions containing the protein of interest were pooled and concentrated using Amicon® Ultra Centrifugal Filter Units with a 3 kDa molecular weight cutoff membrane (Millipore) before size exclusion chromatography on a Superdex200 HiLoad 16/600 (GE Healthcare), which was equilibrated with 25 mM Tris-HCl pH 7.5 and 150 mM NaCl. All purification steps were performed at 4°C, and all buffers were filter-sterilized (0.22 μm). The obtained fractions were analyzed using SDS-PAGE, relevant fractions were pooled (Supplementary Figure S5) and the protein concentration of the final solution was determined using the BCA assay.