Anti his tag antibody

Bacterial whole-cell lysates and SyBV overexpressing S1 were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The blocked membrane was then incubated with anti-His tag antibody (Thermo Fisher Scientific). After incubation with horseradish peroxidase-conjugated secondary antibody, the immunoreactive bands were detected with a chemiluminescent substrate.

We performed binding assays by incubating the indicated amounts of GST-OPN [10 (link)] with the recombinant extracellular domain of CD44v (Lifespan Biosciences, Seattle, WA, USA) in 10 mM HEPES buffer, pH 7.4, plus 150 mM NaCl and 0.05% Tween 20. Heparin or divalent cations were added as indicated. A tube prepared in parallel contained 10% of the input. After 1 h at 4 °C, either GSH-Sepharose was added to pull down GST-OPNa, or nanoCLAMP resin (Nectagen, Kansas City, KS, USA) was added to pull down CD44v via its SUMO-tag. In select experiments, the order was reversed and the pull-down of the target molecule (1 h at 4 °C) preceded the addition of the binding partners. Following another hour of incubation at 4 °C, the resins were washed four times, and the bound fractions were released with reducing SDS-PAGE sample buffer and heating. The eluted proteins were resolved on a 10% polyacrylamide gel and detected via Western blotting with anti-OPN antibody O-17 (IBL America, Minneapolis, MN, USA) or anti-His-tag antibody (ThermoFisher, Waltham, MA, USA) and ECL visualization.

All recombinant proteins used in this study were His-tagged for normalization. SARS-CoV-2 and SARS-CoV N proteins were purified from pcDNA3.1-SARS-CoV-2 N or pDual-GC-SARS-CoV N transfected HEK293 T cells using Ni Sepharose affinity chromatography (GE Healthcare). SARS-CoV-2 S1 and SARS-CoV S1 were obtained from Sino Biological. SARS-CoV-2 RBD and SARS-CoV RBD were custom produced by GenScript. The amino acid sequence of these recombinant proteins are given in Supplementary Table 2. 25 μg of each antigen was coupled onto MagPlex-c microsphere (Luminex) using xMAP antibody coupling kit (Luminex) according to the manufacturer’s instructions. To assess the antibody reactivity, 1250 beads/antigen were incubated with various serum samples at 1:100 dilution in PBS containing 1% BSA for an hour at 37°C with agitation. The bound antibodies were detected with goat anti-human IgG, PE (eBioscience) at 1:1000 dilution. The level of antibody binding was determined using the Luminex MAGPIX system. The readings were normalised by dividing with the readings obtained from the anti-His tag antibody (Thermo Scientific) and presented as net mean fluorescence intensity (MFI).

To challenge cells with cytokines, cells were incubated with Interferon-γ (IFNγ; Cell Signaling Technology 80385S), FasL (AdipoGen AG-40B-0130-3010), TRAIL (R&D Systems 375-TL-010), or TNF-α (AdipoGen AG-40B-0019-3010) for 24 h. TRAIL was crosslinked by incubating with anti-His Tag antibody (Thermo Fisher Scientific MA121315, 1:500) for 15 min at room temperature. Cell viability was measured using CellTiter-Glo (Promega G7571) and protein was harvested for western blots. For evaluating Caspase 8 activity, cells were incubated with FasL or crosslinked TRAIL for 3 h and harvested for Caspase 8 colorimetric assay (R&D Systems K113-100). IFNγ in the cell culture media of the T cell cytotoxic assay was quantified using an ELISA kit (Thermo Fisher Scientific KHC4021).