7 k mwco zeba spin columns

Polyclonal anti-human IL-6 antibodies (AF-206-NA, R&D Systems) were covalently coupled to oligonucleotides. 10 µg of antibodies per conjugated PLA probe at a concentration of 2 µg/µl in PBS was activated by addition of a 33.3-fold molar excess of dibenzyl cyclooctyne NHS ester (DBCO-NHS ester; Jena Bioscience), freshly dissolved in DMSO (Sigma-Aldrich) and incubated at room temperature for 30 min. Thereafter, the activated antibodies were purified from DBCO-NHS using the 7 K MWCO Zeba Spin columns (Thermo Scientific) that had been equilibrated with PBS. The activated antibodies were then split into two aliqotes, mixed with a 2.5-fold molar excess of the respective azide-modified oligonucleotides, and incubated overnight at 4 °C.

Purified recombinant proteins were desalted in kinase buffer using 7K MWCO Zeba Spin Columns (Thermo Scientific) and processed as described in [15 (link)]. Briefly, samples with Aurora1 were incubated at 30 °C, 30 min with 0.1 mM ATP for activation of the kinases. Subsequently, [³²P]ATP and substrates (Arabidopsis histone H3 ~10 µg, common substrate of Aurora1; [25 (link)]) were added and incubated for an additional 60 min at 30 °C.