Question: What are the requirements for antigens for Barcode Enabled Antigen Mapping for BCR discovery (BEAM-Ab)?
Answer: Biotinylated antigens are required for BEAM-Ab, with the following recommendations:
- Antigens should be high purity and quality
- Antigens in the size range of 10 kDa to 200 kDa have been tested in-house
- Up to 15 antigens and one negative control can be tested per BEAM-Ab experiment
- Antigens should be properly biotinylated. If not experienced at antigen biotinylation, it is recommended to use commercial vendors or other researchers that are able to provide high-quality biotinylation services and advice.
- Mono-biotinylated antigens (which can be generated using AviTag™ technology) are a recommended option, although poly-biotinylated antigens can also be used.
- 10x Genomics does not provide protocols or reagents for biotinylation. Our in-house tests were performed on antigens that were biotinylated by commercial vendors. An example of an antigen we tested in-house is biotinylated SARS-CoV-2 Spike protein from ACROBiosystems.
- It is reasonable to expect a turn-around time of 5-7 weeks for the generation of biotinylated antigen, though some vendors have popular or common antigens of interest in various biotinylated forms.
- The quantity of antigen required for each BEAM-Ab experiment will vary based on the antigen molecular weight. Refer to the BEAM-Ab Assembly Workbook (Document CG000597) for calculations.
- For example, an antigen with a molecular mass of 100 kDa and a concentration of 0.2 µg/µl would require 19 µl antigen per BEAM-Ab Assembly. Each BEAM-Ab Assembly is sufficient for labeling up to 5 million cells.
- Before starting with the BEAM-Ab workflow, each antigen must be individually pre-screened by assembling with the BEAM Conjugate, labeling non-experimental samples, and performing flow cytometric analysis. See: How do I pre-screen antigens or peptides in the BEAM assay?
For additional guidance, refer to the BEAM Experimental Planning Guide (CG000596)
Products: Single Cell Immune Profiling, Barcode Enabled Antigen Mapping (BEAM)