Question: What is the purpose of the quenching step in the Barcode Enabled Antigen Mapping (BEAM) assay?
Answer: The quenching step serves to reduce non-specific binding and prevent antigen/peptide swapping when multiple BEAM Assemblies are pooled together. The Quenching Reagent provided in the BEAM Core Kit is used for quenching. The composition of the Quenching Reagent is proprietary.
The quenching steps differ between the BEAM-Ab and BEAM-T assays:
- Quenching is performed using the Quenching Reagent
- Quenching is performed using the Quenching Reagent and the Negative Control Peptide.
- The Negative Control peptide will load into any MHC molecules that have not yet been loaded with a target peptide. Ensuring that all MHC molecules are loaded with a peptide before the BEAM-T Assemblies are pooled together will reduce the chance of peptide swapping between Assemblies.
The quenching step is only performed during the actual BEAM experiment. It is not performed during the antigen/peptide pre-screening protocol. The reasons are outlined below.
BEAM-T - why is the quenching step not performed when pre-screening peptides?
- In BEAM-T, one of the goals of the peptide pre-screening protocol is to check whether the peptide is able to load into the MHC. If cells are labeled with a BEAM-T Assembly generated from a peptide that does not properly load into the MHC, the unloaded/empty MHC leads to a shift in PE fluorescence, with many cells appearing to be PE-positive.
- During the actual BEAM-T experiment we add an excess of Negative Control Peptide during the quenching step. The Negative Control Peptide will load into any empty/unloaded MHC molecules. If we performed the quenching step during pre-screening, this would prevent us from identifying peptides that did not properly load into the MHC.
BEAM-Ab - why is the quenching step not performed when pre-screening antigens?
- When pre-screening individual antigens for BEAM-Ab, a poor quality / sticky antigen typically results in a shift in PE signal, regardless of whether or not the quenching step is performed. We chose to remove the quenching step from the BEAM-Ab pre-screening protocol to simplify the protocol and save 30 min of time.
Products: Single Cell Immune Profiling, Barcode Enabled Antigen Mapping (BEAM)