Minidawn treos

A miniDAWN TREOS (Wyatt Technology Corporation) coupled with an ÄKTA pure system (GE Healthcare) was used for molar mass measurement. The procedure followed the protocol used for analytical gel filtration analysis.

The samples in PBS were injected onto a SEC column (Superdex 200 Increase 10/300, GE Healthcare Life Sciences, Marlborough, MA, USA) at a flow rate of 0.5 mL/min. at 7 °C, detected at 280 nm, and coupled to a multi angle light scattering detector (miniDAWN TREOS, Wyatt, Santa Barbara, CA, USA).

100 μL of protein (either ANKS3-SAM/ANKS6-SAM heterodimer at 15 mg/mL, ANKS6-SAM wt at 10 mg/mL, or ANKS6-SAM R823W at 10 mg/mL) was analysed by SEC-MALS. Protein was loaded onto a WTC-030S5 analytical size-exclusion column (Wyatt Technology Co.) equilibrated in 20 mM Tris pH 8.0, 0.15 M NaCl, (+2 mM BME for the ANKS3-SAM/ANKS6-SAM heterodimer) using an AKTA purifier (GE) and run at 0.5 mL/min on a miniDAWN TREOS (Wyatt Technology Co.). Eluted protein peaks were analysed for calculated molecular weight and monodispersity using ASTRA software (Wyatt Technology Co.)

Purified, non-TEV cleaved STEAP4_EM was injected on a Superdex200 10/300increase column equilibrated in 20 mM Tris pH 8.0, 150 mM NaCl and 0.08% digitonin at a flowrate of 0.75 ml/min. For molecular weight measurements, the column was connected to an online light scattering detector (miniDAWN TREOS, Wyatt Technology) device and a differential refractive index monitor (Shimadzu RID- 10A) on a HPLC system (Shimadzu). Chromatograms were collected and analyzed using the ASTRA software suite (Wyatt). The protein conjugate module was employed for separating the molecular weights of protein and micelle. For STEAP4, a dn/dc of 0.184 ml/g was used based on one predicted N-linked glycan per subunit. For digitonin, we used a previously determined dn/dc value of 0.153 ml/g^{43 (link)}. Bovine serum albumin was used as a standard.

SEC-MALS analysis for molecular weight and oligomer state determination was performed as described [35 (link)]. Briefly, TvCyP2 (1 mg/mL) and TvCyP2-∆N (1 mg/mL) were used for SEC-MALS analysis. The column (Enrich Tm SEC. 70 10 × 300, Bio-Rad Laboratories, Santa Barbara, CA, USA) was used with a flow rate of 0.5 mL/min in the buffer system of 20 mM Bis-Tris, 50 mM NaCl at pH 6.0 and 25 °C. Detectors such as an ultraviolet-visible (UV) detector (QELS, Wyatt Technology, Santa Barbara, CA, USA), a static light-scattering detector (mini DAWN TREOS, Wyatt Technology, Santa Barbara, CA, USA), a quasi-elastic light-scattering detector (QELS, Wyatt Technology, Santa Barbara, CA, USA), and a refractive index detector (Optilab T-rEX, Wyatt Technology, Santa Barbara, CA, USA) all were aligned with the column. Bovine serum albumin (Sigma, A1900, Saint Louis, MO, USA) was used as a standard for calibration and optimization. The molecular weight was calculated by using ASTRA 6 with dn/dc value set to 0.185 mL g^-1.