Protocol to identify endogenous proximal proteins using biotinylation by antibody recognition
Summary: Biotinylation by antibody recognition (BAR) is an antibody-based approach for mapping proximal protein interactions in cells. Here, we present a protocol to biotinylate and identify proximal proteins using BAR. We describe steps for defining proximity labeling reaction conditions, assessing...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-03-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166724007123 |
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| author | Camilla Rega Mercedes Pardo Lesley-Ann Martin Jyoti Choudhary |
| author_facet | Camilla Rega Mercedes Pardo Lesley-Ann Martin Jyoti Choudhary |
| author_sort | Camilla Rega |
| collection | DOAJ |
| description | Summary: Biotinylation by antibody recognition (BAR) is an antibody-based approach for mapping proximal protein interactions in cells. Here, we present a protocol to biotinylate and identify proximal proteins using BAR. We describe steps for defining proximity labeling reaction conditions, assessing enrichment using western blot, and sample preparation for mass spectroscopy analysis. We then detail procedures for data analysis and identifying proximal proteins. This approach differs from standard proximity labeling techniques, which rely on genetically engineered enzymes fused to the target protein.For complete details on the use and execution of this protocol, please refer to Rega et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-4f48858d3f5f429684102b0b9c8af00d |
| institution | Kabale University |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-4f48858d3f5f429684102b0b9c8af00d2025-01-11T06:41:59ZengElsevierSTAR Protocols2666-16672025-03-0161103547Protocol to identify endogenous proximal proteins using biotinylation by antibody recognitionCamilla Rega0Mercedes Pardo1Lesley-Ann Martin2Jyoti Choudhary3The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK; Corresponding authorThe Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKThe Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKThe Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UKSummary: Biotinylation by antibody recognition (BAR) is an antibody-based approach for mapping proximal protein interactions in cells. Here, we present a protocol to biotinylate and identify proximal proteins using BAR. We describe steps for defining proximity labeling reaction conditions, assessing enrichment using western blot, and sample preparation for mass spectroscopy analysis. We then detail procedures for data analysis and identifying proximal proteins. This approach differs from standard proximity labeling techniques, which rely on genetically engineered enzymes fused to the target protein.For complete details on the use and execution of this protocol, please refer to Rega et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166724007123cell biologyprotein biochemistryproteomics |
| spellingShingle | Camilla Rega Mercedes Pardo Lesley-Ann Martin Jyoti Choudhary Protocol to identify endogenous proximal proteins using biotinylation by antibody recognition STAR Protocols cell biology protein biochemistry proteomics |
| title | Protocol to identify endogenous proximal proteins using biotinylation by antibody recognition |
| title_full | Protocol to identify endogenous proximal proteins using biotinylation by antibody recognition |
| title_fullStr | Protocol to identify endogenous proximal proteins using biotinylation by antibody recognition |
| title_full_unstemmed | Protocol to identify endogenous proximal proteins using biotinylation by antibody recognition |
| title_short | Protocol to identify endogenous proximal proteins using biotinylation by antibody recognition |
| title_sort | protocol to identify endogenous proximal proteins using biotinylation by antibody recognition |
| topic | cell biology protein biochemistry proteomics |
| url | http://www.sciencedirect.com/science/article/pii/S2666166724007123 |
| work_keys_str_mv | AT camillarega protocoltoidentifyendogenousproximalproteinsusingbiotinylationbyantibodyrecognition AT mercedespardo protocoltoidentifyendogenousproximalproteinsusingbiotinylationbyantibodyrecognition AT lesleyannmartin protocoltoidentifyendogenousproximalproteinsusingbiotinylationbyantibodyrecognition AT jyotichoudhary protocoltoidentifyendogenousproximalproteinsusingbiotinylationbyantibodyrecognition |