Superdex 75 pc 3.2 30

The hCC WT protein and two of its variants (V57G and V57P) at the concentration of 30 μM (in PBS) were incubated with DPC and SDS membrane mimetics or their mixture, at different mimetic concentrations (1:1, five step dilution series with the highest concentration of C ${}_{DPC:SDS}$ = 5 mM, C ${}_{DPC}$ = 4.2 mM or C ${}_{SDS}$ = 0.8 mM depending on the mimetic used), for 24 h and at different temperatures (22 °C—room temperature, 37 °C—human body temperature). After incubation, 10 L of the mixture was applied on the gel filtration column (Superdex 75 PC 3.2/30, GE Healthcare Life Sciences, USA) and eluted with PBS buffer. The results were analyzed with Chromax 2007 (POL-LAB, Poland) and OriginPro 2018 software to verify if the hCC monomer-dimer equilibrium had changed.

Analysis of intra- and intermolecular structures in the oligonucleotides library created for the determination of the best substrate for TrwC_R was determined by gel filtration in a Superdex75 PC 3.2/30 (GE Healthcare). 2.0 μl of each oligonucleotide (100 μM) were diluted in 18 μl of buffer solution (100 mM Tris pH 7.6, 200 mM NaCl, 10 mM EDTA) and injected in the column loop of 25 μl. The elution buffer was 100 mM Tris 7,6, 200 mM NaCl, 1 mM EDTA. When we analyzed the protein, 20 μl of stock solution (42 nM of TrwC_R) was injected alone. The complexes of TrwC_R with oligonucleotides were loaded after 30 min incubation at room temperature, with an excess of oligonucleotide (ratio 1:1.5) to ensure maximum complex formation.
To study the new balance achieved with Rep-like substrates, IRDye700 labeled oligonucleotide R388 H(14+14) mixed with TrwC_R in presence of 10 mM Mg₂⁺ during 1 h was analyzed by gel filtration and native electrophoresis. After high resolution gel filtration column chromatography of the binding mixture, 10 μl of the fractions were loaded into a 5% non-denaturing acrylamide gel, and the fluorescent label of the oligonucleotide was detected with Odyssey infrared scanner.

Oligonucleotides for site-directed mutagenesis and dNTPs were purchased from Oligo.pl (Warsaw, Poland) or Sigma Aldrich (Poland, Poznan). DNA sequencing was performed at the Laboratory for Nucleic Acid and Protein Detection (Pomeranian Science and Technology Park, Gdynia, Poland). Pfu polymerase was purchased from Fermentas. S-Sepharose, Superdex 75 PC 10/300, Superdex 75 PC 3.2/30 columns and Gel Filtration Low Molecular Weight Calibration Kits were from GE Healthcare (Warsaw, Poland). DNA purification kits were purchased from A&A Biotechnology (Gdansk, Poland). Unless otherwise specified, all reagents were of molecular biology or analytical grade.