# Experimental validation plan — not performed

No laboratory access or experimental results are currently available. This document is a study-design checklist, not a record of completed experiments or a substitute for a laboratory-approved protocol.

## Candidate selection gate

Do not synthesize a candidate merely because it has a short DRAMP match, an SVM score, or a predicted signal peptide. Before committing resources:

1. Resolve host/non-host origin, inspect the complete ORF and local alignment, review possible vector/phage or microbial homology, and deduplicate identical sequences.
2. Establish defensible mature-peptide boundaries. Removing a predicted signal peptide does not automatically identify the final processed AMP.
3. Obtain independent sequence verification from an appropriate biological specimen, if available. Record specimen, tissue and sequence evidence.
4. Review whether a computational paper should simply report unresolved candidates rather than claim a validated ant AMP. There is currently no experimentally validated ant-origin AMP in this project.

## Assay design to arrange with a suitable laboratory

- Confirm synthesized peptide identity, sequence, purity, modifications and solubility; retain certificates and mass-spectrometric confirmation where appropriate.
- Use an approved broth microdilution method and appropriate reference organisms selected by the laboratory for the biological question. Include a concentration series, growth, sterility, solvent and positive-antimicrobial controls, and report endpoint and medium.
- Use independent experimental runs and document technical repeats separately. Predefine exclusions and the analysis plan; retain all raw measurements and controls.
- If antimicrobial activity is supported, characterize selectivity with appropriate mammalian-cell toxicity and hemolysis assays under the laboratory's ethics and biosafety procedures. Absence of predicted toxicity is not evidence of safety.
- Add stability or mechanism studies only when supported by the initial biological results and the eventual manuscript's claims.

## Evidence required before calling validation complete

Candidate identities and mature boundaries; assay methods; organisms/strains; batch and replicate metadata; raw measurements and control performance; analysis scripts; activity and toxicity estimates with uncertainty; limitations and any contradictory findings.

No activity, MIC, toxicity, selectivity, secretion or mechanism result has been generated in this project. Laboratory measurements cannot be supplied by computational analysis.

## Primary guidance

- [EUCAST methodology and instructions](https://www.eucast.org/ast_of_bacteria/mic_determination)
- [CLSI M07 standard overview](https://clsi.org/standards/products/microbiology/documents/m07/)

The laboratory must select the applicable current method; these links do not establish that the procedures have been carried out.