Jul 23, 2025

Public workspaceBinding Assay (His-tagged proteins)

DOI
https://dx.doi.org/10.17504/protocols.io.261gednqdv47/v1
  • Terran Stenger1,
  • Philippa Kennedy1
  • 1University of Minnesota Medical School
Terran D Stenger
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DOI: https://dx.doi.org/10.17504/protocols.io.261gednqdv47/v1
Protocol Citation: Terran Stenger, Philippa Kennedy 2025. Binding Assay (His-tagged proteins). protocols.io https://dx.doi.org/10.17504/protocols.io.261gednqdv47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 01, 2023
Last Modified: July 23, 2025
Protocol Integer ID: 85806
Keywords: binding, binding assay, tag present on the trike construct, flow cytometry, tagged protein, functional characterization of these therapeutic molecule, antigen interaction, 10x polyhistidine, insights into trike, therapeutic molecule, trike construct, assessment of trike, trike
Abstract
To assess the binding specificity of tri-specific killer engagers (TriKEs) to their intended target, we utilize a flow cytometry-based binding assay leveraging the 10x polyhistidine (His) tag present on the TriKE construct, as previously described by our lab (see references). This assay enables assessment of TriKE binding efficiency and specificity, providing insights into TriKE-antigen interactions that inform further optimization and functional characterization of these therapeutic molecules.
Fig 1. Schematic of the binding assay (figure made in BioRender).

Guidelines
Processed tissue additional controls:
Fc Receptor Blocking Solution, cat. 422302, BioLegend, (1/100 dilution)
Materials
  • R10: RPMI (Gibco Cat. No. 2240-089) + 10% fetal bovine serum (Gibco Cat. No. 26140079) + 100 U/mL Penicillin and Streptomycin (Gibco Cat. No. 15140122)
  • Flow buffer: 1% human AB serum, 0.5 mM EDTA in PBS
  • PE anti-His tag antibody, cat. 362603, Biolegend
  • PE mouse IgG2A Isotype control, cat. 400212, Biolegend
  • 2% paraformaldehyde/PBS
Troubleshooting
Cell Preparation
Cells are resuspended in R10 at 1.5 million cells/mL, and Amount200 µL (300k cells) are plated in each well in a U bottom 96 well plate.
Centrifigation300 x g, 00:05:00 The plate is spun in a centrifuge at 300g for Duration00:05:00 (this is approximately 1200rpm in a large benchtop centrifuge). The supernatant is removed.

5m
Incubation with His-Tagged Protein
30m
Amount50 µL of 4X histidine(His)-tagged treatments are added to the pellet.
Recommended final concentrations: 0.3 nM, 3 nM, 30 nM for TriKEs and 3 nM, 30 nM and 300 nM for TriKE-PACC.
Prepare 4X drug in R10.
Amount150 µL of R10 is added. Resuspend.
Incubate for Duration00:30:00 at 37°C.
30m
After the incubation, samples are washed twice with flow buffer to remove excess, unbound protein.
Extracellular + Live/Dead Staining
30m
Cells are stained in Amount50 µL of flow buffer (1% AB serum, 0.5 mM EDTA in PBS) containing:
  • Amount.05 µL LIVE/DEAD Fixable Near-IR Dead Cell Stain, cat. L34976, ThermoFisher
  • Amount5 µL PE anti-His Tag Antibody, cat. 362603, Biolegend

Note: As controls, untreated cells are stained with the anti-His antibody, and TriKE-treated cells are stained with an isotype control antibody.
Incubate for Duration00:20:00 at 4°C. After the incubation, samples are washed twice with flow buffer to remove excess antibody.
20m
Cells are resuspended in Amount100 µL of 2% paraformaldehyde/PBS and incubated at room temperature in the dark for Duration00:10:00 to fix them. Afterwards, wells are topped up to Amount200 µL with flow buffer and washed once.
10m
Flow Cytometry
Samples are analyzed via flow cytometry.

Fig. 2: Example gating strategy in FlowJo

Protocol references
1.) Felices M, Lenvik TR, Davis ZB, Miller JS, Vallera DA. Generation of BiKEs and TriKEs to Improve NK Cell-Mediated Targeting of Tumor Cells. Methods Mol Biol. 2016;1441:333-46. doi: 10.1007/978-1-4939-3684-7_28. PMID: 27177679; PMCID: PMC5823010.

2.) Phung SK, Zorko NA, Soignier Y, Waller RL, Shackelford M, Walker JT, Nelson TD, Selleck C, Bendzick LE, Kotz LE, Kile QM, Bozicevich AJ, Miller SE, Khaw M, Shetty M, Hinderlie P, Ehrhardt M, Li Y, Luo X, Dehm SM, Antonarakis ES, Kennedy PR, Miller JS, Felices M. A PSMA-Targeted Tri-Specific Killer Engager Enhances NK Cell Cytotoxicity against Prostate Cancer. Cancer Immunol Res. 2025 Feb 3;13(2):258-272. doi: 10.1158/2326-6066.CIR-24-0273. PMID: 39545924; PMCID: PMC11790377.