Strep tactin sepharose resin

To validate the interaction pairs by Co‐IP, HEK293 cells (5 × 10⁵ per well) in 6‐well plate were co‐transfected with Strep‐HA‐tagged (500 ng) prey and V5‐tagged (500 ng) bait constructs using Fugene 6 transfection reagent (Promega). After 24 h of transfection, cells were rinsed with ice‐cold PBS and lysed with 1 ml HENN lysis buffer per well (50 mM HEPES pH8.0, 5 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.5% IGEPAL, 1 mM DTT, 1 mM PMSF, 1.5 mM Na₃VO₄, 1 × Protease inhibitor cocktail) on ice. The cell lysate was collected, and a clear supernatant was obtained by centrifugation (16,000 g, 20 min, 4°C). 30 µl of Strep‐Tactin® Sepharose® resin (50% suspension, IBA Lifesciences GmbH) was washed in a microcentrifuge tube twice with 200 µl HENN lysis buffer (4,000 g, 1 min, 4°C). The clear lysate was added to the pre‐washed Strep‐Tactin beads and incubated for 1 h on a rotation wheel at 4°C. After incubation, the beads were collected by centrifugation and washed three times with 1 ml HENN lysis buffer (4,000 g, 30 s, 4°C). After the last wash, 60 µl of 2 × Laemmli sample buffer (Bio‐Rad, 1610737) was added directly to the beads and boiled at 95°C for 5 min. Samples were later used for immunodetection via dot‐blot.

HEK293 cells were seeded into six-well plates at a density of 5 × 10⁵ per well and incubated overnight. The cells were cotransfected with Strep-HA-tagged (500 ng) NFIA, NFIB or NFIC and one V5-tagged (500 ng) prey construct using Fugene 6 transfection reagent (Promega). Twenty-four hours after transfection, cells were harvested, washed with ice-cold PBS and lysed with 1 ml HENN lysis buffer per well (50 mM HEPES pH 8.0, 5 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.5% IGEPAL, 1 mM DTT, 1 mM PMSF, 1.5 mM Na3VO4, 1× protease inhibitor cocktail) on ice. The cell lysate was collected, and protein extracts were collected following centrifugation at 16,000 × g for 20 min at 4 °C. Meanwhile, 40 µl of Strep-Tactin® Sepharose® resin (50% suspension, IBA Lifesciences GmbH) was washed in a microcentrifuge tube twice with 200 µl lysis buffer (4000 × g, 1 min, 4 °C). The clear cell lysate was added to the prewashed Strep-Tactin beads and incubated for 1 h on a rotation wheel at 4 °C. After incubation, the beads were collected by centrifugation and washed three times with 1 ml lysis buffer (4000 × g, 30 sec, 4 °C). After the last wash step, 50 µl of 2× Laemmli sample buffer (Bio–Rad, 1610737) was added onto the beads, and the bound complexes were eluted by boiling for 5 min at 95 °C, followed by dot blot analysis.

To obtain a homogeneous nucleotide load, the nucleotide-free ObgE-Strep protein was incubated (on ice) for 1 h with 1 mM of the nucleotide before starting the experiment. ObgE-strep was mixed with the His₆-tagged YbiB protein in a 1:3 molar ratio (final concentrations of 10 and 30 μM, respectively) in binding buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 5 mM MgCl₂, 5 mM β- mercaptoethanol, 5% glycerol), either with or without 100 μM of the corresponding nucleotide. Samples containing either only ObgE-strep or YbiB were diluted in binding buffer to the corresponding concentration and served as controls. The samples were incubated on ice for 1 h and an ‘input’ sample was taken before incubating with Strep-Tactin^® Sepharose^® resin (iba lifesciences) on ice for 30 min. Each sample was centrifuged at 1000 × g for 1 min at 4°C and the supernatant served as unbound fraction (‘flow-through’). Subsequently, the beads were washed six times with 120 μl binding buffer, collecting the supernatant as wash fractions. The proteins were eluted by incubating the beads on ice for 5 min with 100 μl elution buffer (binding buffer containing 2.5 mM desthiobiotin), followed by centrifugation at 1000 × g for 1 min at 4°C. The supernatant was kept as elution fraction. Finally, the different fractions (input, flow-through, wash and elution) were analyzed on SDS-PAGE.

SPRTN-SSH was expressed in HEK293 cells for 24h. Following treatment with FA (1 mM for 1h), cells were washed twice with ice-cold PBS, and cell pellet was lysed with 1 mL of ice-cold IP lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, 10 mM NEM, protease and phosphatase inhibitors) containing 250 U/ml of benzonase. After complete digestion, lysates were cleared at 500 g for 10 minutes. Extracts were quantified, normalized, and brought to volume. Proteins were denatured with 1% SDS at 55°C for 10 minutes (total volume 1 ml). Samples were diluted 10X with 50 mM Tris HCl pH 7.4, 150 mM NaCl, 1% Triton X-100. SPRTN-SSH was captured in a 10 mL volume with Strep-Tactin®Sepharose® resin (IBA lifesciences) for 3h at 4°C. Beads were washes 3 times with 50 mM Tris HCl pH 7.4, 150 mM NaCl, 0.05% Triton X-100, and bound proteins eluted in 50 μL 2X Laemmli buffer.

To validate the interaction pairs by Co‐IP, HEK293 cells (5 × 10⁵ per well) in 6‐well plate were co‐transfected with Strep‐HA‐tagged (500 ng) bait and V5‐tagged (500 ng) prey constructs using Fugene 6 transfection reagent (Promega). After 24 h of transfection, cells were rinsed with ice‐cold PBS and lysed with 1 ml HENN lysis buffer per well (50 mM HEPES pH 8.0, 5 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.5% IGEPAL, 1 mM DTT, 1 mM PMSF, 1.5 mM Na₃VO₄, 1 × Protease inhibitor cocktail) on ice. The cell lysate was collected, and a clear supernatant was obtained by centrifugation (16,000 × g, 20 min, 4 °C). 30 µl of Strep‐Tactin® Sepharose® resin (50% suspension, IBA Lifesciences GmbH) was washed in a microcentrifuge tube twice with 200 µl HENN lysis buffer (4000 × g, 1 min, 4 °C). The clear lysate was added to the pre‐washed Strep‐Tactin beads and incubated for 1 h on a rotation wheel at 4 °C. After incubation, the beads were collected by centrifugation and washed three times with 1 ml HENN lysis buffer (4000 × g, 30 s, 4 °C). After the last wash, 60 µl of 2 × Laemmli sample buffer (Bio‐Rad, 1610737) was added directly to the beads and boiled at 95 °C for 5 min. Samples were later used for immunodetection via dot‐blot.

WT cells expressing Dri1-Strep-tag II, SdhB1-FLAG protein fusions, or untagged proteins were grown in liquid BG−11 at 30 °C and shaken at 90 rpm in an Innova® 44/44 R shaker (New Brunswick) for 6 days. Cell lysis was performed as mentioned above with the addition of 150 mM NaCl. The protein concentrations of the soluble fractions were normalized by Bradford assay (BioRad) for protein pull-downs. A total of 20 µg of soluble proteins were incubated with 10 µL of Strep-Tactin® Sepharose® resin (IBA-LifeSciences) at 24 °C for 2 hours with or without 200 µM hemin. To remove protein unspecific binding, the resin was washed five times with 100 mM Tris-HCl, 150 mM NaCl, 20% glycerol, and 1 mM EDTA pH 8.0. Proteins were eluted with 20 µL of 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and 2.5 mM desthiobiotin, pH 8.0, boiled for 5 min with SDS-sample buffer, and separated by SDS-PAGE (4-20% ExpressPlus™ PAGE Gel, Genscript), followed by immunoblotting.

DvFdhAB was expressed and affinity-purified from Desulfovibrio vulgaris Hildenborough, as previously described [2 (link)]. The DvFdhAB containing a Strep-tag was produced by homologous recombination and purified by affinity chromatography using a Strep-Tactin^® Sepharose^® resin (IBA Lifesciences). After protein purification, the sample buffer was exchanged and the samples were stored in 20 mM Tris-HCl buffer with 10% glycerol and 10 mM NaNO₃, pH = 7.6. Routinely, protein concentration was determined based on UV/Vis ( ${\epsilon }_{410 \mathrm{nm}}=43.45 {\mathrm{mM}}^{-1}{\mathrm{cm}}^{-1}$ ) and the purity of samples was judged by 12% SDS-polyacrylamide gel [2 (link)]. The samples were stored at −80 °C until further use.