Superdex 75 increase 10 300

SEC–MALS–SAXS data sets were collected using the 18-ID-D BioCAT Beamline at the Advanced Proton Source (APS) at Argonne National Laboratory (Chicago, IL). Samples were centrifuged for 5 min at 13,000 rpm to remove any potential aggregates prior to column loading. Samples containing 4–9 mg/mL of GRB2 WT or mutants in 250 μL were injected onto a 24 mL Superdex 75 Increase 10/300 analytical-grade column (GE) equilibrated with 20 mM Tris pH 8.0, 150 mM NaCl, and 1 mM DTT at a flow rate of 0.5 mL/minute on an Agilent 1300 chromatography system. Column eluant was analyzed in line by the UV absorbance detector of the Agilent 1300 chromatography system, then subsequently directed into the DAWN Heleos-II light scattering (LS) and OptiLab T-rEX refractive index detectors in series. Finally, the elution trajectory directed samples into a 1.0 mm ID quartz capillary SAXS sample cell. Scattering data were collected every 1 s using a 0.5 s exposure and detected with a Pilatus 3 1 M pixel detector (DECTRIS) with a 12 keV (1.033 Å wavelength) X-ray beam covering a q-range of 0.0045 < q < 0.35 Å − 1 (q = 4π/λsinθ, where λ is the wavelength and 2θ is the scattering angle). Accurate protein molecular weights from MALS data were determined using the ASTRA software (Wyatt Technology).

The Apn5 protein was subjected to size-exclusion chromatography (Superdex-75 Increase 10/300; GE Healthcare). The technology support for SEC was provided by Yin P lab in Huazhong Agricultural University following the relative reference^{42 (link)}. The buffer contained 25 mM Tris-HCl, pH 8.0, and 150 mM NaCl. The flow rate was set as 0.5 ml min⁻¹. All the fractions were collected as 0.5 ml per tube for further SDS–PAGE and inhibition activity assay. The relative amount of flagellin was determined by comparing the target protein band volume by using software Quantity One (Bio-Rad, USA). The relative amount of subtilomycin was determined by inhibition activity assay.

The protein complexes between Rsu-1 and the PINCH-1 LIM4-5 domains for crystallization experiments were prepared by incubating each purified protein in a 1:1 M ratio by a rotor at 4 °C for at least 2 h. The protein complex mixtures were loaded onto a size-exclusion chromatography column of either Superdex 200 10/300 GL (GE Healthcare) or Superdex 75 Increase 10/300 (GE Healthcare) pre-equilibrated in a buffer consisting of 20 mM Tris, pH 7.5, and 150 mM NaCl. The major peak fractions containing the target protein complex were pooled and concentrated with Vivaspin 20 (MWCO 10K) centrifugal concentrator for crystallization experiments. The protein concentration was quantitated by measuring absorbance at 280 nm with NanoDrop 2000c Spectrophotometer from Thermo Fisher Scientific.