Rapid and Efficient Isolation of Total RNA-Bound Proteomes by Liquid Emulsion-Assisted Purification of RNA-Bound Protein (LEAP-RBP).
Bio Protoc
; 14(14): e5236, 2024 Jul 20.
Article
en En
| MEDLINE
| ID: mdl-39100593
ABSTRACT
The critical roles of RNA-binding proteins (RBPs) in all aspects of RNA biology fostered the development of methods utilizing ultraviolet (UV) crosslinking and method-specific RNA enrichment steps for proteome-wide identification and assessment of RBP function. Despite the substantial contributions of these UV-based RNA-centric methods to our understanding of RNA-protein interaction networks, their utility is constrained by biases in RBP recovery and significant noise contributions, which can confound meaningful interpretation. To overcome these issues, we recently developed a method termed Liquid Emulsion-Assisted Purification of RNA-Bound Protein (LEAP-RBP) and introduced quantitative signal-to-noise (SN)-based metrics for the proteome-wide identification of RNA interactomes and accurate assessment of global RBP occupancy dynamics. Compared to existing methodologies, LEAP-RBP provides significant advantages in speed, cost, efficiency, and selectivity for RNA-bound proteins. In this work, we provide a step-by-step guide for the successful application of the LEAP-RBP method for both small- and large-scale investigations of RNA-bound proteomes. Key features ⢠Unbiased and efficient isolation of total RNA-bound protein, RNA, and protein from biological samples. ⢠Cost-effective identification of proteome-wide RNA interactomes and validation of direct RNA-binding protein functionality. ⢠Robust and accurate assessment of context- and/or condition-dependent RBP occupancy state dynamics.
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1
Colección:
01-internacional
Base de datos:
MEDLINE
Idioma:
En
Revista:
Bio Protoc
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos