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Consequently FRET serves as a powerful spectroscopic ruler for probing molecular interactions. Whilst cell based FRET assays report bulk relative changes in FRET efficiency in a population, single molecule FRET (smFRET) is capable of deconvoluting these population averages into distinct structural states. However, the lack of universal benchmarks prevents the direct translation of in vitro distance measurements to the intracellular environment and vice versa. Here, we present a modular protein ladder designed to harmonize FRET data across diverse platforms. Using an engineered repeating TPR motif and self\u2010labeling enzymes, we demonstrate that our standards yield consistent FRET efficiencies across expression systems (mammalian and bacterial) and labeling strategies (self labeling enzymes and click chemistry with non\u2010canonical amino acids). By providing a predictable calibration curve, the ladder enables interpolation between different experimental FRET modalities, including confocal smFRET, flow cytometry based\u2010FRET and Fluorescence Lifetime Imaging Microscopy FRET (FLIM\u2010FRET). This is the necessary infrastructure to relate molecular distances from the test tube to the\u00a0cell.<\/jats:p>","DOI":"10.1002\/advs.76672","type":"journal-article","created":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T12:08:42Z","timestamp":1784635722000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Universal Protein Ladder for Standardization of Diverse FRET Assays"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9872-233X","authenticated-orcid":false,"given":"Evelyn R.","family":"Smith","sequence":"first","affiliation":[{"name":"Division of Neuroscience, School of Medicine and Population Health, Faculty of Health, Sheffield Institute for Translational Neuroscience The University of Sheffield Sheffield UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5148-4469","authenticated-orcid":false,"given":"Katie L.","family":"Gelder","sequence":"additional","affiliation":[{"name":"Molecular and Cellular Biology, School of Biosciences, Faculty of Science The University of Sheffield Sheffield UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lewis","family":"Hunter\u2010Craig","sequence":"additional","affiliation":[{"name":"Division of Neuroscience, School of Medicine and Population Health, Faculty of Health, Sheffield Institute for Translational Neuroscience The University of Sheffield Sheffield UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0276-6486","authenticated-orcid":false,"given":"Daniel A.","family":"Bose","sequence":"additional","affiliation":[{"name":"Molecular and Cellular Biology, School of Biosciences, Faculty of Science The University of Sheffield Sheffield UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timothy D.","family":"Craggs","sequence":"additional","affiliation":[{"name":"School of Mathematical and Physical Sciences, Faculty of Science The University of Sheffield UK"},{"name":"Exciting Instruments Sheffield UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1796-1508","authenticated-orcid":false,"given":"Alison E.","family":"Twelvetrees","sequence":"additional","affiliation":[{"name":"Division of Neuroscience, School of Medicine and Population Health, Faculty of Health, Sheffield Institute for Translational Neuroscience The University of Sheffield Sheffield UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,7,21]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.60416"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1002\/9783527656028"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi101184g"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1364\/ECBO.2007.6633_57"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.aan1133"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2222"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz797"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2026.114875"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2818.2010.03437.x"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1529\/biophysj.104.054114"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-3495(03)75126-1"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-018-0193-x"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-023-01807-0"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0408164102"},{"key":"e_1_2_10_16_1","doi-asserted-by":"crossref","unstructured":"P.Huter S.Arenz L. 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