Akta pure fplc

SAXS was performed at BioCAT (beamline 18ID at the Advanced Photon Source, Chicago) with in-line size-exclusion chromatography (SEC-SAXS) to separate sample from aggregates and other contaminants thus ensuring optimal sample quality. The sample was loaded onto a Superdex 75 10/300 Increase column (Cytiva), which was run at 0.6 ml/min by an AKTA Pure FPLC (GE), and the eluate, after it passed through the UV monitor, was flown through the SAXS flow cell. The flow cell consists of a 1.0 mm ID quartz capillary with ~20 μm walls. A coflowing buffer sheath is used to separate sample from the capillary walls, helping prevent radiation damage^{62 (link)}. Scattering intensity was recorded using an Eiger2 XE 9 M (Dectris) detector which was placed 3.688 m from the sample giving us access to a q-range of 0.027 Å⁻¹ to 0.42 Å⁻¹. 0.5 s exposures were acquired every 1 s during elution and data were reduced using BioXTAS RAW 2.1.4^{63 (link)}. Buffer blanks were created by averaging regions flanking the elution peak and subtracted from exposures selected from the elution peak to create the I(q) vs q curves used for subsequent analyses. 3D electron density reconstruction was done using DENSS^{64 (link)} incorporated in RAW 2.1.4.

SAXS data were collected at the BioCAT beamline 18-ID at the Advanced Photon Source. In-line size exclusion chromatography was performed using a Superdex 200 Increase 10/300 GL column, which was run at 0.7 ml/min by an AKTA Pure FPLC instrument (GE Healthcare Life Sciences). The eluate was passed through the UV monitor and through a 100 μL quartz flow cell and exposed to the X-ray beam every 2 s with 0.5 s exposures. Data were collected at room temperature using a wavelength equal to 1.033 Å, a Pilatus3 1M detector (Dectris), and a sample-to-detector distance equal to 3.5 m. Buffer subtraction was performed using BioXTAS RAW [27 ]. PRIMUS and AUTORG were used to calculate R_g values [28 ,29 ], and GNOM was used to generate D_max values and pair-wise distribution plots [29 ,30 ].

Purified human ARHGAP18 was produced by bacterial expression of an N-terminal-SUMO-HIS-tagged protein purified using a NiNTA resin. The SUMO tag was cleaved using purified ULP1 and then the ARHGAP18 was further purified using a HiLoad 16/600 superdex 200 size-exclusion chromatography installed on a General Electric AKTA pure FPLC. For antibody production, this purified protein was exchanged into PBS and denatured by adding 1 mM DTT and then boiled for 5 min at a final concentration of 2.1 mg/mL before flash freezing in liquid nitrogen. The frozen ARHGAP18 antigen was then shipped to Pocono Rabbit Farm & Laboratory, Inc (Canadensis, PA) for antibody production in rabbits. ARHGAP18 antibody specificity was confirmed by western blot at a concentration of (1:1000) against the antigen, the ARHGAP18^-/- cells, and observation of a band shift when expressing a GFP-tagged variant of ARHGAP18 in Jeg3 cells (Figure 2C). The animal use protocol approved by Cornell University was IACUC number 2014-0109 to A. Bretscher.