Minidawn treos instrument

SEC-MALS was used to determine the monodispersity and molecular weight of P0ct in solution. Chromatography was performed using an Äkta Purifier (GE Healthcare) and a Superdex 75 pg 10/300GL (GE Healthcare) column with 20 mM HEPES, 300 mM NaCl, pH 7.5 as mobile phase. A 250-µg P0ct sample was injected into the column at an isocratic flow of 0.4 ml/min, and light scattering recorded using a Wyatt miniDAWN TREOS instrument. The UV signal recorded at 280 nm was used as concentration source using the extinction coefficient of P0ct (Abs 0.1% = 1.061) calculated using ProtParam⁵¹ . Data were analyzed using the ASTRA software (Wyatt).
Full length P0 was similarly run on SEC-MALS in the presence of different detergent micelles. The running buffer contained 250 mM sodium phosphate (pH 8.0), 1 mM EDTA, and 0.1% of either DPC or LDAO. A Superdex 200 column was used in the experiment. The protein conjugate analysis routine in the ASTRA software was used to obtain the molecular weight of P0 and the detergent in the separated peaks.

SEC-MALS was used to analyze protein monodispersities and molecular weights. SEC was performed using an Äkta Purifier (GE Healthcare) and a Superdex 75 Increase 10/300 GL column (GE Healthcare) in 20 mm HEPES, pH 8.5, 100 mm NaCl, 2 mm EDTA. For each measurement, 200–300 μg of protein was injected and gel filtrated at a flow rate of 0.5 ml/min. Light scattering was recorded using a miniDAWN TREOS instrument (Wyatt Technology). Protein concentration in each elution peak was determined using differential refractive index. The data were analyzed using the ASTRA 6.2 software (Wyatt Technology).

Potential protein aggregation was measured via size-exclusion chromatography (SEC) coupled with multi-angle laser light scattering (MALLS). Protein samples were injected onto a Superdex 200 Increase10/300 GL column (GE Healthcare), with PBS pH 7.4 as running buffer at 0.5 ml/min, coupled to an inline ultraviolet-detector (Shimadzu), a multi-angle light scattering miniDAWN TREOS instrument (Wyatt) and an Optilab T-rEXrefractometer (Wyatt) at 25°C. A refractive index increment (dn/dc) value of 0.185 ml/g was used for protein concentration and molecular mass determination. Data were analyzed using the ASTRA6 software (Wyatt). Correction for band broadening was applied using parameters derived from BSA injected under identical running conditions. For the analysis of IL-33traps, conjugate analysis was performed using theoretical protein extinction coefficients and a dn/dc value of 0.160 ml/g for the glycan modifier.