If you know a protein interacts with a receptor or a target but not which part of it does the work, synthetic peptides give you a direct way to find out. Cut the sequence into pieces, test which pieces bind, then change residues one at a time until binding fails. This hub sets out that workflow end to end: how to plan the peptide set, which assay to measure it with, how to get from a region to individual residues, and which controls decide whether the answer is believable.
| Stage | Peptide set | What you learn |
|---|---|---|
| 1. Localize | Overlapping peptides spanning the sequence | Which region or regions carry binding activity |
| 2. Minimize | Truncations from both ends of the active region | The shortest peptide that still binds |
| 3. Resolve | Alanine or systematic substitutions across the minimal peptide | Which individual side chains the interaction depends on |
| 4. Confirm | Scrambled controls, non-binding variants, a labelled version for a second assay format | That the signal is sequence-specific and not an artifact of the format |
An overlapping scan divides the sequence into peptides of fixed length that each start a fixed number of residues after the last. Two numbers define it: the peptide length and the step size, which is the offset between consecutive peptides. The overlap is the difference between them.
A useful shortcut is to scan selectively rather than exhaustively. If structural data, sequence conservation or a prior deletion experiment already points at part of the protein, a dense scan of that region plus a coarse scan of the rest costs far less than uniform coverage. We can build either as a peptide library, in individual vials or in an array format.
What a linear scan can and cannot find
An overlapping scan finds binding determinants that lie in one continuous stretch of sequence. It will miss a site assembled from two regions that are far apart in sequence but adjacent in the folded protein, and it will miss a site that requires a specific conformation the short peptide cannot adopt. A negative scan result is therefore weaker evidence than a positive one. The same distinction is set out for antibodies in linear and conformational epitopes, and the conformational limits of short peptides in how do peptides fold.
Once a region is identified, truncation defines its boundaries and substitution defines its content. Truncating from each end in steps of one or two residues shows where activity drops off, which usually gives a minimal peptide several residues shorter than the original hit.
Substitution comes next. Replacing each position in turn with alanine removes the side chain while leaving the backbone, so a loss of binding points to a side chain that contributes directly. Glycine substitution tests backbone flexibility, and substituting with a residue of similar size but different chemistry separates shape from charge or hydrogen bonding. Our alanine scanning page covers the formats, and epitope mapping and peptide scanning covers ordering the sets.
For dozens to hundreds of peptides, a plate-based or array format gives a yes-or-no answer cheaply. Treat the output as a ranking that tells you what to follow up, not as affinity data.
For the handful of peptides that matter, a label-free kinetic method such as SPR gives association and dissociation rates as well as affinity, and reveals weak binders that endpoint assays miss.
Which end of the peptide is tethered can decide whether it binds at all. A biotin or label at the wrong terminus can block the determinant. See designing peptides for binding studies.
Mapping sets commonly need biotinylated and fluorescently labelled versions, usually with a spacer between label and sequence.
Send us the protein sequence and the region you want covered, with your preferred peptide length and step size if you have one, and we will propose a peptide set and price it. If you are not sure how to scope the first round, tell us the assay you plan to use and we will suggest a design. Email sales@lifetein.com or use the quotation form.
Work in rounds. Scan the sequence with overlapping peptides to localize the region, truncate from both ends to find the minimal peptide, substitute each position with alanine to identify the critical residues, then confirm with scrambled and non-binding controls in a second assay format.
Peptides of twelve to twenty residues are usual. Step size sets resolution and cost: scanning a 300-residue protein with 15-mers at a step of five needs about sixty peptides, while a step of three needs about a hundred.
A linear scan only finds determinants contained in one continuous stretch of sequence. Sites assembled from distant regions of the fold, sites that need a specific conformation, and sites that depend on a post-translational modification will all be missed.
That is normal. A fragment of a binding surface loses the surrounding contacts and the conformational restraint the intact protein provided, so it typically binds far more weakly.
A scrambled peptide of the same composition, a non-binding variant from the alanine scan, confirmation in a second assay format, competition against the full-length interaction, and known net peptide content for any quantitative comparison.
Research use only. LifeTein peptides and proteins are supplied for laboratory research use. They are not for human or veterinary use, and not for diagnostic or therapeutic use. See our research use policy.