Protino ni nta resin

To analyze interactions between Strep-tagged TatA and PspA variants, co-elution experiments were carried out as described previously [23 (link)], with the exception that cell debris was removed prior to TatA-strep purification. For co-elution assays with His-tagged proteins, cells were resuspended in 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM Imidazol, and disrupted via French Press, and affinity chromatography was carried out using the Protino Ni-NTA resin (Macherey-Nagel, Düren, Germany) with the above buffer system. After loading and 6 washing steps (each 1 column volume), His-tagged proteins were eluted by increasing the imidazole concentration to 250 mM. Elution fractions were analyzed via SDS-Page and Western blotting.

For purification of soluble proteins, 10 mL of soluble BugBuster™ protein extract was mixed with 1 mL of Protino^® Ni-NTA resin (Macherey-Nagel, Düren, Germany). The mixture was stirred slowly on a turn-over shaker for 1 h at 4 °C to let the target fusion proteins bind to the matrix. Thereafter, the resin was filled in a column and the excess fluid was allowed to pass through the filter by gravity. The resin was then washed using 20 bed volumes (~10 mL) of washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 50 mM imidazole, pH 8.0). Subsequently, the resin was mixed with 0.1 mL of elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0), following incubation at room temperature for 30 min. The first eluate was collected and 0.1 mL of elution buffer was added, followed by incubation over night at 4 °C. The eluate was then collected and the elution step was repeated twice to ensure quantitative elution. The purity and quantity of collected eluates were analyzed by SDS-PAGE and western blot analysis. The elution fractions were pooled and the protein concentration was determined by Bradford protein assay, as per the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). The purified proteins were stored at 4 °C.