Geldoc instrument

Protein samples were mixed with Tris/glycerol native sample buffer and loaded onto a 4–12% polyacrylamide gel. The gel was run in Tris/glycine buffer supplemented with 0.02% Coomassie Brilliant Blue G-250 dye until the samples had migrated 1 cm into the gel, then without the dye at 20 mA for 2 hours. Bovine serum albumin (BSA Fraction V, Sigma) was used as a reference for molecular weight. The samples were then transferred onto a 0.45 μm nitrocellulose membrane (Amersham Protran, GE Healthcare) for 2 hours at 200 mA in a transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3). The membrane was quenched by using 5% non-fat milk in TBS-Tween (50 mM Tris, 150 mM NaCl, 0.02% Tween 20, pH 7.6) supplemented with 0.1% BSA overnight at 4°C. The blot was then incubated with Anti-B4GalT1 antibody (HPA010807, Sigma,1:2000) in TBS-Tween + 0.1% BSA for 2 hours at room temperature, washed 3 x 10 min with TBS-Tween before incubation with the goat anti-rabbit HRP 1:10000 (Abliance, Compiègne, France) in TBS-Tween + 0.1% BSA for 1 hour at room temperature. After final washings (4 x 15 min in TBS-Tween), ECL reagent (BioRad) was added and the membrane was photographed using a GelDoc instrument (BioRad). A linear correlation between the log of the molecular weight of BSA oligomers and their migration distance was used to estimate the molecular weights of oligomeric species of B4GalT1.

84bp long genomic DNA around the polymorphism was amplified using specific primers designed using BLAT (59 (link)) (UCSC genome browser, Supplemental Table 3). Amplicons were digested by DdeI (Thermo Fisher Scientific) for 4h at 37°C. Samples were run on a TBE 20% polyacrylamide gel (Invitrogen) at 100V for 3.5h and stained with SYBR Green I nucleic acid gel stain (Thermo Fisher Scientific) in TE buffer (10mM Tris, 1mM EDTA, pH8) for 40 min while shaking. Images were taken on a Biorad Geldoc instrument.

Solubility determination was assessed as previously described (Fleckenstein et al. 2015 (link)). Briefly, 10 µg of CS in 50 mM sodium phosphate buffer pH 7.0 were incubated for 1 h at 45 °C in the presence or absence of HSP1, HSP2 or HSP3 or with a mixture of the three sHSP, at a molar ratio of 16:1 (sHSP:CS). Samples were centrifuged at 20,800 × g for 10 min at 4 °C to separate the soluble fraction from the pellet (i.e., containing aggregated proteins). Proteins in the soluble fraction were precipitated by cold acetone (10 min on ice) and centrifuged at 14,000 × rpm, 30 min at 5 °C. The precipitated proteins and the pellet were solubilised by the Laemmli buffer (Bio-rad) and resolved on a hand-casting 12% TGX Stain-Free polyacrylamide gel (Bio-rad), further stained with colloidal Coomassie G-250. Gel images were acquired by Gel Doc instrument (Bio-rad) and quantified by Image software. Ten µg of CS (0.3 μM in 50 mM sodium phosphate buffer pH 7.0), processed as all holdase assay samples, were used as control.