Thermo Scientific Pierce Magnetic RNA-Protein Precipitation Kit provides researchers with a simplified, robust method to use end-labeling RNA serves as bait to enrich for protein-RNA interactions.

Features of the Magnetic RNA-Protein Sedimentation Kit:

•Direct – uses end-labeled RNA to directly capture ribonucleoprotein complexes; no or no Antibodies required
•Easy to use – No centrifuge cups or centrifugation required to enrich RBPs; simplified procedure requiring minimal hands-on time (less than 3 hours) after RNA labeling reaction
•Flexible – Use in vitro transcribed RNA or synthetic RNA for labeling of various lengths and complexities; use endogenous, overexpression, and in vitro< /i>Proteins successfully enriched in translation lysates
•Specificity – Magnetic beads have low background noise; irrelevant RNA or mutated RNA does not significantly affect the enrichment of specific RBPs
•< b>Economical – Lower price than purchasing commercially synthesized end-labeled RNA, magnetic beads and reagents separately
•Complete Set – Contains labeling module and enrichment module with detection in the module Required buffers; including positive control RNA, negative control RNA, and RBP antibody

Magnetic RNA-Protein Precipitation Kit is provided for use desthiobiotin terminally labeled Reagents for efficient RNA enrichment of RNA-binding proteins (RBPs) and streptavidin magnetic beads. The complete kit contains sufficient reagents for 20 RNA labeling reactions and 20 protein-RNA sedimentation assays. This method of direct enrichment of protein-RNA interactions can serve as an alternative to antibody capture of protein-RNA complexes or nucleic acid-embedded proteins. Another advantage of this kit is that it provides validated controls for both the labeling reaction and the sedimentation analysis. The kit is suitable for a variety of downstream applications, including Western blotting and mass spectrometry (MS).

Includes:
The complete kit includes Pierce RNA 3'-end desthiobiotinylation kit, positive and negative RNA controls, and nucleic acid-compatible streptomycin. Hosin Magnetic Beads and RBP Enrichment and Elution Buffer

Requires:
User-provided RNA and lysate (experimental sample)

Applications:
• Mutation analysis
• Structure-function analysis
• Identification of RNA-protein interactions
• MS analysis of unknown RNA:protein binding pairs or complexes

Using labeled RNA as bait to enrich RNA-binding proteins is superior to antibody enrichment because of its direct capture of interactions. Included in this kit is Pierce RNA 3'-Terminal Desthiobiotinylation Kit, which uses T4 RNA Ligase joins a single cytidine diphosphate nucleotide to the 3'-end of single-stranded RNA. Desthiobiotin can be used as both a detection target and an elutable affinity handle. The length of the spacer arm between the nucleotide and desthiobiotin has been optimized to efficiently attach to the beads without hindering the binding of RNA to protein.

The procedure for RNA binding protein enrichment has been optimized and easy to use. Labeled RNA is first captured onto microbeads to prepare for protein binding. Equilibrate the RNA-bound beads in protein-RNA binding buffer, then add protein lysate. After washing, samples are eluted using either nondenaturing biotin elution buffer or SDS-PAGE loading buffer. The eluted sample is ready for a variety of downstream operations, including Western blotting or mass spectrometry analysis.

Control systems for sedimentation assays include the 3'-untranslated region of androgen receptor (AR) RNA, poly(A)25 RNA, and mammalian cell lysates. The proximal 3'-untranslated region (UTR) of the androgen receptor RNA contains multiple UC-rich regions that bind HuR as well as Poly(C)-binding proteins (CP1 and CP2). These RNA-binding proteins regulate mRNA stability (HuR) as well as mRNA metabolism and translation (CP1 and CP2). Negative control RNA and poly(A)25 RNA do not contain binding sites for HuR or poly(C) BP.

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