Goat anti mouse igg antibody hrp conjugate

Eluted fetuin-binding fraction (50 μg in 50 μg of 2× Laemmli loading buffer (Sigma-Aldrich)) and MDBK-bound protein fractions (resuspended in 100 μg 1× Laemmli) were electrophoresed in NuPAGE 4–12% Bis-Tris 10-well gels (Invitrogen, California, USA) and stained with Coomassie blue (BioRad, Hertfordshire, UK). Proteins were transferred from gels to nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK) by semi-dry blotting following the manufacturer’s protocols (Invitrogen), and membranes were blocked in 5% (w/v) non-fat milk (Bio-Rad) overnight. Membranes were incubated for 1 h with mouse anti-histidine tag antibody (1/1000; Merck Millipore, Hertfordshire, UK), then washed three times with tris buffered saline-Tween20 (TBS-Tween) 0.05% (v/v) and incubated for 1 h with goat anti-mouse IgG antibody HRP conjugate (1/5000; Merck Millipore). Membranes were washed three times with TBS-Tween 0.05% (v/v), once with TBS and finally with distilled water before adding Luminata substrate (Merck Millipore). Chemiluminiscence was visualised in a G:BOX (Syngene, Cambridge, UK) and images were taken with GeneSnap 7.12 software (Syngene).

Cells were lysed in cold radio immunoprecipitation assay buffer (Life Technologies). The cell lysate was pre-cleaned with protein-G sepharose (GE Healthcare, catalogue no. 17-0618-02) for 30 min and subjected to immunoprecipitation with each indicated antibodies (1 μg), incubated for 1 h on ice and then 25 μl of protein-G sepharose beads were added for additional 4–6 h. The beads were then washed for four times, boiled with 15 ml of 2 × SDS sample buffer for 5 min and the proteins were separated on 10% SDS–PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% fat-free dried milk in Tris-buffered saline with 0.5% Tween 20 (TBST) for 2 h. The membranes were then incubated in appropriate primary antibodies overnight at 4 °C. Membranes were washed in TBST and then incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies (EMD Millipore Corp, goat anti-rabbit IgG antibody, HRP conjugate, catalogue no. 12–348; goat anti-mouse IgG antibody, HRP conjugate, catalogue no. 12–349). Membranes were washed in TBST, and the signals were visualized using enhanced chemiluminescence substrate (Thermo Scientific, Waltham, MA) and quantified using the Bio-Rad Image software. Representative uncropped blots are shown in Supplementary Fig. 9.