Flow-TriCEPS
for flow cytometry
Cellular responses to ligands such as peptides, proteins, pharmaceutical drugs or entire pathogens are generally mediated through interactions with proteins expressed at the cell surface. The LRC-TriCEPS kit is designed to directly identify the target proteins of your ligand on the living cells directly using near-physiological conditions.
Dualsystems now offers the pretest TriCEPS experiments as services or as a kit.
The pretest test is designed to screen different cell types and decide which cells to use later for the identification of the unknown targets of your ligand of interest in the LRC-TriCEPS main experiment.
In case functional assays are available TriCEPS coupled ligands can also be tested to see if a similar output can be achieved with a TriCEPS coupled ligand compared to a ligand that is not coupled to TriCEPS.
As first step, the TriCEPS v.2.0 molecule is coupled to the ligand of interest (peptide, protein, Antibody, ADC or other primary amine containing molecules) and to the positive control ligand (e.g. transferrin) and negative control ligand (e.g. glycine). This coupling reaction is tested with Dot blot to assess if the coupling worked.
Then, the TriCEPS coupled ligands are added to the non-oxidized cells to assess whether the ligand binds to the unknown targets at the cell surface and TriCEPS does not interfere with the ligand receptor interaction.
Flow Cytometry tests with TriCEPS coupled ligand on oxidized cells can be further performed to test cell viability and ligand target binding as it will occur in the main LRC-TriCEPS experiment.
After successful completion of all pretests, the main experiment can be planned and carried out as described in the LRC-TriCEPS user manual choosing one of the service options provided by Dualsystems Biotech AG.
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Using Flow-TriCEPS with flow cytometry
Ligand of interest and controls are coupled to TriCEPS. Coupling of TriCEPS to the ligand can be controlled by dot blot for protein ligands. Cells expressing the unknown targets are treated with TriCEPS coupled ligand and binding is visualized using a fluorophore.
Example of a Flow-TriCEPS experiment
Competition experiment using Flow-TriCEPS. MDA-MB-231 cells are treated with the TriCEPS coupled ligand (orange). In order to confirm that the observed binding is due to the binding of transferrin to its target on the cells, cells are treated with different amounts of unlabeled transferrin before adding TriCEPS coupled ligand. The more unlabeled transferrin is added the smaller the shift of TriCEPS-transferrin is observed since the unlabeled transferrin competes with the TriCEPS coupled transferrin for the binding to its receptor.
TriCEPS-Transferrin on MDA cells
Flow TriCEPS-Kit flyer
Ligand-receptor capture LRC-TriCEPSflyer
(Figure 1) Flow-TriCEPS is coupled to transferrin (positive control), glycine (negative control) and the ligand of interest and added on MDA-MB-231 breast cancer cells. Coupling efficiency is controlled by dot blot (Figure 2). The mean fluorescent shift is compared across the different ligands. It is expected the ligand of interest gives a larger shift than glycine. If different parameters are tested (time, temperature, pH, etc.) the condition leading to the largest shift for the ligand of interest is used for the main identification experiment.
Customers Testimonials – LRC-TriCEPS Service
Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.
LRC-TriCEPS customers worldwide
Over 200 satisfied customers from 28 countries.
LRC-TriCEPS publications worldwide
LRC-TriCEPS / HATRIC-LRC Publications
Concerning the LRC-TriCEPS or HATRIC-LRC platforms.
Acidity changes immunology: a new VISTA pathway
Nature Article VISTA interaction with PSGL-1 identified by LRC-TriCEPS
Anti-VISTA antibody that inhibits Vista function and blocks interaction with PSGL-1 and VSIG3 proteins slows tumor growth
Phage resistance at the cost of virulence
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008032 October 7, 2019
White Paper Ligand-Receptor Identification Methodologies Details Matter
Validation of extracellular ligand–receptor interactions by Flow‑TriCEPS
Cardiac Targeting Peptide, a Novel Cardiac Vector: Studies in Bio-Distribution, Imaging Application, and Mechanism of Transduction
Leukocyte differentiation by histidine-rich glycoprotein/stanniocalcin-2 complex regulates murine glioma growth through modulation of anti-tumor immunity
Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions
HATRIC-based identification of receptors for orphan ligands
Staphylococcal Superantigens Use LAMA2 as a Coreceptor GPCT signaling To Activate T Cells
Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma
Phenotypic screening—the fast track to novel antibody discovery
Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology
Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5
Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization
Identification of cell surface receptors for the novel adipokine CTRP3
Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing
- Received:
- Accepted:
- Published online:
A Mass Spectrometric-Derived Cell Surface Protein Atlas
Protter
Ligand-based receptor identification on living cells and tissues using TRICEPS
Flex your TRICEPS