Ktapure fplc system

SEC-MALS analysis was performed as previously described^{26 (link)}. Briefly, WT Drp1 and mutants at the indicated injection concentrations were sieved through a Superose 6 10/300GL column attached to an ÄKTApure FPLC system (Cytiva) connected in line with DAWN Heleos-II 18-angle MALS and Optilab T-rEX differential refractive index (dRI) detectors from Wyatt Technology. Full-length GIPC-1 (10 μM at injection) was sieved using a Superdex 200 10/300 GL column similarly. Data were analyzed using the ASTRA 7 software also from Wyatt Technology.

The suspension with the harvested cells was defrosted and 90 µl of DNAse was added (∼2 µg ml⁻¹). The cell suspension was sonicated using a Fisherbrand Model 705 Sonic Dismembrator in three 2 min cycles. The disrupted cells were centrifuged (4°C and 14 000 rev min⁻¹ for 1 h). The protein was then filtered and loaded onto a BioLogic DuoFlow FPLC system (Bio-Rad, USA) equilibrated in buffer A (10 mM Tris, 50 mM sodium formate, 10 mM imidazole pH 8.5) and buffer B (10 mM Tris, 50 mM sodium formate, 500 mM imidazole pH 8.5). The protein was purified by metal-affinity chromatography using a nickel-charged column at a flow rate of 1 ml min⁻¹. The protein was eluted with an increasing gradient of imidazole (0%, 10%, 60% and 100% buffer B; DmmarA was eluted in the 10% and 60% gradients). The purified protein from the 60% imidazole gradient was concentrated to ∼5 ml using Amicon Ultra-15 Centrifugal Filter Units (10 kDa cutoff).
5 ml of protein was loaded onto an ÄKTApure FPLC system (Cytiva, Sweden) equilibrated in gel-filtration buffer (10 mM Tris, 50 mM sodium formate pH 8.5). The protein was purified by size-exclusion chromatography using a Superdex 16/60 200 pg column (GE Healthcare, UK). SDS–PAGE was used to check the purity of fractions from affinity chromatography and size-exclusion chromatography (run at 200 V and 400 mA for 40 min).

The suspension with the harvested cells was defrosted and 90 µl of DNAse was added (∼2 µg ml⁻¹). The cell suspension was sonicated using a Fisherbrand Model 705 Sonic Dismembrator in three 2 min cycles. The disrupted cells were centrifuged (4°C and 14 000 rev min⁻¹ for 1 h). The protein was then filtered and loaded onto a BioLogic DuoFlow FPLC system (Bio-Rad, USA) equilibrated in buffer A (10 mM Tris, 50 mM sodium formate, 10 mM imidazole pH 8.5) and buffer B (10 mM Tris, 50 mM sodium formate, 500 mM imidazole pH 8.5). The protein was purified by metal-affinity chromatography using a nickel-charged column at a flow rate of 1 ml min⁻¹. The protein was eluted with an increasing gradient of imidazole (0%, 10%, 60% and 100% buffer B; DmmarA was eluted in the 10% and 60% gradients). The purified protein from the 60% imidazole gradient was concentrated to ∼5 ml using Amicon Ultra-15 Centrifugal Filter Units (10 kDa cutoff).
5 ml of protein was loaded onto an ÄKTApure FPLC system (Cytiva, Sweden) equilibrated in gel-filtration buffer (10 mM Tris, 50 mM sodium formate pH 8.5). The protein was purified by size-exclusion chromatography using a Superdex 16/60 200 pg column (GE Healthcare, UK). SDS–PAGE was used to check the purity of fractions from affinity chromatography and size-exclusion chromatography (run at 200 V and 400 mA for 40 min).