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Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification
While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein contents often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity western blots on single cells (scWesterns) are attractive since they...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10312704/ https://www.ncbi.nlm.nih.gov/pubmed/37398364 http://dx.doi.org/10.1101/2023.06.13.544857 |
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author | Long, Mariia Alibekova Benman, William Petrikas, Nathan Bugaj, Lukasz J. Hughes, Alex J. |
author_facet | Long, Mariia Alibekova Benman, William Petrikas, Nathan Bugaj, Lukasz J. Hughes, Alex J. |
author_sort | Long, Mariia Alibekova |
collection | PubMed |
description | While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein contents often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity western blots on single cells (scWesterns) are attractive since they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 10(3) molecules, a 520-fold improvement. This enables us to detect 85% and 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies respectively, compared to 47% of cells using in-gel detection. These results suggest compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagents — not previously accessible for in-gel use — for further signal amplification and detection of low abundance targets. |
format | Online Article Text |
id | pubmed-10312704 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Cold Spring Harbor Laboratory |
record_format | MEDLINE/PubMed |
spelling | pubmed-103127042023-07-01 Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification Long, Mariia Alibekova Benman, William Petrikas, Nathan Bugaj, Lukasz J. Hughes, Alex J. bioRxiv Article While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein contents often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity western blots on single cells (scWesterns) are attractive since they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 10(3) molecules, a 520-fold improvement. This enables us to detect 85% and 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies respectively, compared to 47% of cells using in-gel detection. These results suggest compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagents — not previously accessible for in-gel use — for further signal amplification and detection of low abundance targets. Cold Spring Harbor Laboratory 2023-06-14 /pmc/articles/PMC10312704/ /pubmed/37398364 http://dx.doi.org/10.1101/2023.06.13.544857 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator. |
spellingShingle | Article Long, Mariia Alibekova Benman, William Petrikas, Nathan Bugaj, Lukasz J. Hughes, Alex J. Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification |
title | Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification |
title_full | Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification |
title_fullStr | Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification |
title_full_unstemmed | Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification |
title_short | Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification |
title_sort | enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10312704/ https://www.ncbi.nlm.nih.gov/pubmed/37398364 http://dx.doi.org/10.1101/2023.06.13.544857 |
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