Mouse anti his tag antibody

Total soluble protein was quantified by Bradford assay and analyzed based on SDS-PAGE according to Laemmli 1970 (link) with 6% stacking gel, and 15% separating gel and run at a constant current of 2.2 mA/cm. Samples were mixed with an equal volume of 2X sample loading buffer and treated by heating at 95 °C for 5 min in sample loading buffer before loading on gels. SDS–PAGE gels were stained with Coomassie blue R250. For western blot analysis, the separated polypeptides in acrylamide gel were transferred to nitrocellulose membrane and stained with the appropriate antibody mouse anti-his tag antibody, (Biolegend, 652,501) with a dilution ratio of 1:1000 and horseradish peroxidase-conjugated anti-mouse immunoglobulin G antibody (Sigma) with a dilution ratio of 1:2000. Finally, 3,3’- Diaminobenzidine (Sigma) was used to visualize the blots.

Cell lines were resuspended in FACS buffer (0.5% BSA/0.1% NaN₃ in PBS) and incubated with various antibody constructs at 2 μg/mL for 20 min at 4 °C. hIgG1 binding to target cells was assessed using a PE-conjugated goat anti-human antibody (12–4998-82, Thermo Fisher). Pro- and mature- forms of GrB-TRA bound constructs were also detected using a mouse anti-His tag antibody (652,502, Biolegend) followed by a PE-conjugated goat anti-mouse reagent (115–115-164, Jackson ImmunoResearch).
To determine caspase 3/7 activity, 3 × 10⁵ target cells were seeded in complete media in a 6-well plate and cultured for 8 h. Non-adherent cells and media were removed and replaced with fresh complete media containing antibody constructs at various concentrations to generate proportionate, non-total cytotoxic responses. Following overnight incubation at 37 °C with 5% CO₂, cell media and adherent cells were collected and incubated with Cell Event Caspase-3/7 Green Detection Reagent and SYTOX AADvanced Dead Cell Stain (Thermo Fisher) per the manufacturer’s instructions.
Flow cytometric analysis was performed on cells using a BD LSR Fortessa (BD Biosciences) and FlowJo v 10.8.1 software (BD Life Sciences).