Typical Sample Requirements 3D Epitope Mapping

ATEM Structural Discovery

4 min read

Typical sample requirements for 3D epitope mapping with cryo-EM

Summary

Before starting an epitope mapping project, most teams ask the same question: what do I need to provide? This article outlines the antigen and antibody samples required for ATEM’s high-throughput 3D epitope mapping platform, explains the workflow your material moves through, and highlights the structural insights that can be generated, with up to five structures delivered in as few as three weeks.

Why choose cryo-EM epitome mapping?

Most epitope mapping methods involve compromise. Peptide- and sequence-based assays are fast, but they often miss conformational epitopes and provide limited information about the true binding interface [1]. X-ray crystallography offers high structural detail but can be challenging.

Cryo-EM avoids many of these limitations. By imaging intact antigen–antibody complexes in near-native conditions, it captures both linear and conformational epitopes without requiring crystallization, making it well suited to a broad range of therapeutic targets.

ATEM’s differentiator is throughput. Rather than solving structures one candidate at a time, ATEM can screen lead series containing tens of candidates in parallel and return up to five structures in roughly three weeks. This allows teams to compare multiple binders early in discovery and make more informed lead-selection decisions. The resulting structural data can also serve as an experimental benchmark for validating and refining AI-generated predictions.

Sample requirements

Here’s what you’ll need before starting a cryo-EM epitome mapping project with us.

Antigen / target protein

Soluble protein or solubilized membrane protein
≥95% purity (by SDS gel)
≥1 mg for initial structure (,5 mg for an additional structure)
≥2 mg/mL concentration
All host species and glycosylation states accepted
Most buffer compositions and pH values accepted
Untagged proteins or small tags (e.g., His, Flag) accepted [2]

Antibody / ligand

IgG*, Fab, VHH, scFv, peptides, aptamers
≥95% purity (by SDS gel)
0.5–1 mg per structure
≥2 mg/mL concentration [2]

* Note: if you are supplying IgGs, we’d prefer 1 mg due to the loss on the IgG to Fab digestion.

Complex

Stable complex formation
≥65 kDa stable molecular mass [2]

Two practical considerations are worth noting. First, full IgGs can be difficult to resolve completely because of their inherent flexibility, so Fabs are often preferred. If only IgG material is available, ATEM can perform Fab isolation. Second, stability is usually more important than size. Transient or heterogeneous complexes are among the most common reasons additional optimization is required.

Material Sourcing

Two options are available:

Option 1 – The source materials are provided by you

In this case, you provide the source materials and we can manage the full shipment process for you, including handling all the required documents, booking the preferred pick-up times, setting up a specialized carrier (with all required packaging, cooling agents and temperature), and tracking the shipping. You only need to drop off the samples.

Option 2 – We source the antigens from established partners

In case you don’t have the antigens, we can source them from different established partners with whom we have great experience. This will require about 3 – 6 weeks, depending on the type of partner you select. If you prefer, we are also happy to engage with your existing partner and make the arrangements with them.

In case you opt for this option, we will need to know the antigen specifics, especially the sequence. As the sequences are highly critical IP, we work with generic antibody templates up to the intermediate structure and only request the specific sequence during the high-resolution 3D structure determination.

What happens after you submit?

Your samples move through our workflow: the antigen–antibody complex is formed and assessed, vitrified for cryo-EM imaging, and reconstructed in three dimensions using single-particle analysis. ATEM begins with a pilot phase to confirm sample suitability, followed by optional biochemical optimization (when needed), and then intermediate- or high-resolution mapping depending on project goals.

Results delivered within weeks

Structural data answers questions that traditional binding assays cannot, such as:

Potential binding site competition
Conformation or oligomerization target changes upon antibody binding.

These insights help teams select stronger leads, and validate computational predictions with experimental evidence.

Intermediate-resolution mapping (4–10 Å) supports lead selection and competitive epitope binning, while high-resolution structures (typically below 4 Å) can provide sidechain-level detail for paratope mapping, mechanism-of-action studies, lead optimization, patent support, and regulatory filings.

In addition to epitope identification, cryo-EM can reveal domain-level binding, steric competition between antibodies, interaction stoichiometry, antigen oligomerization state, and conformational changes induced by binding, providing a more complete picture of how a therapeutic candidate engages its target.

Learn more about our High-throughput 3D Epitope Mapping service

Conclusion

Ensuring samples meet these requirements upfront reduces optimization time and increases the likelihood of obtaining clear, interpretable structural data. If you are considering 3D epitope mapping for your antibody research project, ATEM’s high-throughput cryo-EM platform is designed to deliver structural insight at scale.

Talk with our cryo-EM experts to discuss your samples and project goals.