Dawn heleos mals detector

SEC-MALS was performed at room temperature and using PBS buffer (pH 7.4). Proteins concentrated at 25 µM were loaded on a Hiload 10/300 Superdex 200 pg column (GE Healthcare). The elution of the proteins was monitored with a Dawn Heleos MALS detector (Wyatt Technology). Molecular weight of the proteins was calculated using ASTRA V software.

SEC experiments were performed on 100 µl injections of 70 µM ScMlp1_1–325 with a Superdex 200 (10/300) GL column (GE Healthcare) at 0.5 mL min⁻¹ at 25 °C in 500 mM NaCl, 20 mM HEPES–NaOH (pH 7.5), and 0.5 mM TCEP. Absolute molecular weights were determined using MALS. The scattered light intensity of the column eluent was recorded at 18 different angles using a DAWN-HELEOS MALS detector (Wyatt Technology Corp.) operating at 658 nm after calibration with the monomer fraction of Type V BSA (Sigma). Protein concentration of the eluent was determined using an in-line Optilab T-rex interferometric refractometer (Wyatt Technology Corp.). The weight-averaged molecular weight of species within defined chromatographic peaks was calculated using the ASTRA software version 6.0 (Wyatt Technology Corp.), by construction of Debye plots (KC/Rθ versus sin²[θ/2]) at 1-s data intervals. The weight-averaged molecular weight was then calculated at each point of the chromatographic trace from the Debye plot intercept, and an overall average molecular weight was calculated by averaging across the peak.

Gel filtrated pure MIER1(aa:171–350):HDAC1 complex was reapplied to a Superdex S200 column and monitored with an Optilab T-rEX differential Refractive index detector connected to a DAWN HELEOS MALS detector (Wyatt Technology). The mass of the complex was calculated using ASTRA software version 6.1.

Average MW and size distribution of porphyran were measured by SEC-MALS using an Optilab rEX Refractive Index detector and a Dawn Heleos MALS detector (Wyatt Technology). The separation of the molecules was performed using a Thermo Ultimate 3000 chromatographic system and 3 Shodex SB-805-HQ, SB-804-HQ, and SB-803-HQ columns connected in series. Elution was performed in 0.1 M LiNO₃ with 300 μg/l NaN₃ at 0.5 ml/min. One hundred microliters (100 μl) of 0.1% (w/v) porphyran solution was injected on the system after filtration on a 0.45 μm membrane. Data were processed using the Astra software (Wyatt Technology), using a dn/dc ratio of 0.146 as suggested by the detector supplier.

Glycerol gradient centrifugation with (GraFix) or without fixation was performed similarly to Kastner et al. (52 (link)). Briefly, proteins were sedimented at 40,000 RPM for ∼16 h in a 5 to 30% glycerol gradient at 4 °C using a Beckman SW 60 Ti rotor. Gradients were prepared in 20 mM Hepes (pH 7.5), 0.2 M NaCl, 5 mM MgCl₂, and 1 mM EGTA using a Gradient Master device (BioComp Instruments). For experiments with crosslinking, 0.125% (v/v) glutaraldehyde was added to the 30% glycerol solution before preparing the gradient. Crosslinking was quenched by the addition of 40 mM glycine–HCl (pH 7.5). A Piston Gradient Fractionator (BioComp Instruments) was used for fractionation.
Glycerol in samples was removed by quick dialysis, followed by centrifugal concentration. For mass determination, samples were injected into a TSKgel SuperSW2000 column (Tosoh Corporation) before entering a DAWN HELEOS MALS detector and an Optilab rEX refractive index detector (Wyatt Technology Corporation). The Astra software (Wyatt Technology Corporation) was used to calculate molecular masses.

14-3-3θ (100 μl at 4 mg mL⁻¹) was pre-incubated for 2 h at 20 °C with either the singly-phosphorylated Site 2 peptide at 1:2 molar ratio (one 14-3-3θ dimer per two Site 2 peptides) or the doubly-phosphorylated Sites 2,4 peptide at 1:1 molar ratio (one 14-3-3θ dimer per one Sites 2,4 peptide). The complexes were loaded onto a TSKgel SuperSW2000 column (Tosoh Corporation, Tokyo, Japan) connected in-line to a DAWN HELEOS MALS detector and an Optilab rEX (Wyatt Technology Corporation, Santa Barbara, CA) refractive index detector for mass analysis. Molecular masses were calculated with the program Astra (Wyatt Technology Corporation).