Minidawn treos multi angle light scattering

SEC-MALS experiments were performed using a Superdex Increase 200 10/300 GL column (GE Healthcare) on an Agilent 1260 HPLC Infinity II in PBS buffer at RT (~297 K). Protein elution was monitored by three detectors in series namely, an Agilent multi-wavelength absorbance detector (absorbance at 280 nm and 254 nm), a Wyatt miniDAWN TREOS multiangle light scattering (MALS) detector, and a Wyatt Optilab rEX differential refractive index (dRI) detector. The column was pre-equilibrated overnight in the running buffer to obtain stable baseline signals from the detectors before data collection. Molar mass, elution concentration, and mass distributions of the samples were calculated using the ASTRA 7.1.3 software (Wyatt Technology). A BSA solution (2–4 mg/ml), purchased from Sigma-Aldrich and directly used without further purification, was used to calibrate inter-detector delay volumes, band broadening corrections, and light-scattering detector normalization using standard protocols within ASTRA 7.1.3.

SEC/MALS analysis was performed on an HPLC system (Agilent) coupled to mini DAWN TREOS multi-angle light scattering and RefractoMax 520 refractive index detectors (Wyatt Technology). RNAP-δ-HelD complex was assembled as for cryoEM. 60 μl of the sample at 1–1.3 mg/ml were chromatographed on a Superose 6 Increase 10/300 column (GE Healthcare) in buffer H or buffer H plus 0.15% (w/v) n-octylglucoside, supplemented with 0.02% (w/v) NaN₃, at 18 °C with a flow rate of 0.6 ml/min. Data were analyzed with the ASTRA 6.1 software (Wyatt Technology) using monomeric bovine serum albumin (Sigma-Aldrich) as a reference.

SEC-MALS experiments
were performed using a Superdex Increase 200
10/300 GL column (GE Healthcare) on an Agilent 1260 HPLC Infinity
II in phosphate buffer (PBS or 50 mM KPi KCl pH 7.4) at RT (ca. 297
K). Protein elution was monitored by three detectors in series namely,
an Agilent multiwavelength absorbance detector (absorbance at 280
and 254 nm), a Wyatt miniDAWN TREOS multiangle light scattering (MALS)
detector, and a Wyatt Optilab rEX differential refractive index (dRI)
detector. The column was pre-equilibrated overnight in running buffer
to obtain stable baseline signals from the detectors before data collection.
Molar mass, elution concentration, and mass distributions of the samples
were calculated using the ASTRA 7.1.3 software (Wyatt Technology).
A BSA solution (2–4 mg/mL), purchased from Sigma-Aldrich and
directly used without further purification, was used to calibrate
interdetector delay volumes, band broadening corrections, and light-scattering
detector normalization using standard protocols within ASTRA 7.1.3.

Size exclusion chromatography/multi-angle light scattering analyses were performed on an HPLC system (Agilent) coupled to a mini DAWN TREOS multi-angle light scattering and RefractoMax 520 refractive index detectors (Wyatt Technology). 60 μl (15 nmol) of SuhB were passed over a Superdex 200 increase 10/300 column (GE Healthcare) in 20 mM HEPES, pH 7.5, 50 mM NaCl, 0.02% (w/v) NaN₃ at a flowrate of 0.6 ml/min. Data were analyzed with the ASTRA 6.1 software (Wyatt Technology) using monomeric bovine serum albumin (Sigma-Aldrich) as a reference.