Question: What are the requirements for peptides for Barcode Enabled Antigen Mapping (BEAM) for TCR discovery (BEAM-T)?
Answer: For BEAM-T, peptides should meet the following requirements:
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Peptide should be 9-12 amino acids long.
- Up to 15 peptides and one negative control can be tested per BEAM-T experiment.
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Peptides should be of high quality and purity (>90%).
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Associated QC steps such as Fast Protein Liquid Chromatography (FPLC) or mass spectrometry can be requested from the vendors to ensure the peptide purity as well to confirm the sequence.
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Peptides should have good solubility. Peptide solution should be properly checked to see any indication of poor solubility such as precipitation or cloudiness.
- The quantity of peptide required for each BEAM-T experiment depends on the number of cells used for labeling. Resuspend lyophilized peptide in DMSO to 10 mM, then dilute to 110 µM using sterile PBS. Use 5 µl peptide per BEAM-T Assembly for every 1 million cells.
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Use appropriate MHC monomer-matched peptides.
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Select a peptide that is compatible with the MHC/HLA type of the donor and choose the appropriate 10x Genomics Monomer kit. Prediction engines such as NetMHCpan can be used to predict peptides with potential for binding the specific MHC Class I alleles that will be used in the BEAM-T experiment.
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The lead time to procure peptides could be 3-5 weeks, although more common peptides (various virus peptides for EBV, Flu, or CMV) for the most common MHC types are sometimes in stock with certain vendors.
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Each peptide must be individually pre-screened by assembling with the BEAM Conjugates, labeling non-experimental samples, and performing flow cytometric analysis. See: How do I pre-screen antigens or peptides in the BEAM assay?
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When performing the BEAM-T experiment, include a Negative Control Assembly prepared with the Negative Control peptide provided in each 10x Genomics MHC Monomer kit.
- Peptides should NOT be biotinylated. In the BEAM-T assay, peptides are loaded into a biotinylated MHC monomer. The biotinylated MHC monomer will bind to the streptavidin on the BEAM Conjugate during reagent assembly. The peptide itself should not be biotinylated.
For additional guidance, refer to the BEAM Experimental Planning Guide (CG000596).
Products: Single Cell Immune Profiling, Barcode Enabled Antigen Mapping (BEAM)