Labeling of His-tagged PCNA and ClpB proteins

His-tagged human PCNA protein was purchased from Sigma Aldrich (catalogue number SRP5117), at a concentration of 6.6 µM. The protein was specifically labeled via the histidine linker on the N-terminal side of the protein with the dye ATTO647N containing a Ni-NTA functional group which was also purchased from Sigma Aldrich (catalogue number 02175–250 UG-F). First, the protein was desalted to a labeling buffer (25 mM HEPES, 25 mM KCl, pH 7.8) using a 7 K MWCO Zeba desalting column (ThermoFischer, cat. 89882) and then reacted with dyes at a ratio of 1:4 for 2 hr at RT. The protein was then desalted from the excess of dyes using the same desalting column. The labeling efficiency was estimated using an absorption spectrometer (Nanodrop 2000, ThermoFischer) confirming ~100% labeling efficiency. SDS-page and native gel indicated that the protein is indeed assembled and labeled. N-terminal His-tagged ClpB protein, mutated at residue 428 from alanine to cysteine, was purified as described in a previous publication (Mazal et al., 2019 (link)). The labeling of the single cysteine was done following the same procedure as describe above with ATTO647N maleimide as a specific labeling agent of the cysteine. Absorption spectroscopy, SDS-page and native gel indicate complete assembly of the protein, see previous publication (Mazal et al., 2019 (link)).