Ni sepharose histrap hp

For purification of tritium‐labelled complement 5a ([³H]‐C5a^CF), reaction and feeding mixes were pooled and applied to a 1 mL HisTrap FF column (GE Healthcare) equilibrated in buffer C^C5a using a peristaltic pump. The column was washed with 4 CV buffer C1^C5a, 4 CV buffer C2^C5a and eluted in 4 CV buffer D^C5a. Tritium‐labelled Endothelin‐1 ([³H]‐fET‐1^CF) was purified accordingly with the following modifications: the peptide was purified in buffer C^ET−1 using 0.5 mL bed volume Ni Sepharose HisTrap HP (GE Healthcare) in a gravity flow column. Buffer exchange to buffers C^C5a or C^ET−1 was performed by repeated concentration and dilution in a 3 kDa MWCO concentrator (Amicon) and protein concentration was calculated using the specific activity of incorporated [³H]‐Pro or [³H]‐Phe residues. [³H]‐C5a^CF contains three proline residues, furin‐cleaved [³H]‐pET‐1^CF contains one phenylalanine residue.

Following cross-linking of 200 OD units of decoated spores, frozen pellets were lyophilized and broken as described above. Broken spores were suspended in Urea Binding Buffer (8 M Urea, 500 mM NaCl, 50 mM Tris-HCl, 30 mM imidazole, pH 7.5) and incubated at 4 °C for 2 h. The samples were centrifuged at 6,800 x g for 10 min, and the soluble fraction was collected, filtered, and loaded onto a 1 mL Ni Sepharose HisTrap HP (GE Healthcare) column equilibrated in Urea Binding Buffer. Bound YpeB-His6 was eluted with Urea Elution Buffer (8 M Urea, 500 mM NaCl, 50 mM Tris-HCl, 1 M imidazole, pH 7.5). Fractions were stored at − 80 °C for western blot analysis.