The Thermo Scientific Pierce Magnetic RNA-Protein Pull-Down Kit provides researchers with a streamlined, robust method to enrich protein-RNA interactions using end-labeled RNA as the bait.
The Magnetic RNA-Protein Pull-Down Kit provides reagents to efficiently enrich RNA Binding Proteins (RBPs) using RNA end-labeled with desthiobiotin and streptavidin magnetic beads. The complete kit contains sufficient reagents for 20 RNA labeling reactions and 20 protein-RNA pull-down assays. This direct enrichment of the protein-RNA interaction provides an alternative to antibody capture of protein-RNA complexes or moieties incorporated into the nucleic acid. An added advantage to the kit is that it includes validated controls for both the labeling and pull-down assay. The kit is amenable to several downstream applications, including Western blotting and Mass Spectrometry (MS).
- Direct – capture ribonucleoprotein complexes directly with end-labeled RNA; does not use or require antibodies for pull-down
- Easy to use – no spin cups or centrifugation necessary for the enrichment of RBP; procedure streamlined for 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 complexity; proteins successfully enriched using endogenous, over-expressed, and in vitro translated lysates
- Specific – low bead background; unrelated RNA or mutated RNA does not significantly enrich specified RBPs
- Economical – less expensive than purchasing commercially synthesized end-labeled RNA, magnetic beads, and reagents separately
- Complete – contains both labeling and enrichment modules with buffers necessary for assay; positive control RNA, negative control RNA, and RBP antibody included
Complete kit contains the Pierce RNA 3'-End Desthiobiotinylation Kit, positive and negative RNA controls, nucleic-acid compatible streptavidin magnetic beads, and buffers for RBP enrichment and elution
User-supplied RNA and lysate (experimental sample)
- Mutational analysis
- Structure function analysis
- Identification of RNA:Protein interactions
- MS analysis of unknown RNA:protein binding pairs or complexes
Utilizing labeled RNA as bait for RNA-binding protein enrichment is advantageous over antibody enrichment in that it captures the interaction directly. Included in this kit is the Pierce RNA 3’-End Desthiobiotinylation Kit, which uses T4 RNA ligase to attach a single cytidine bisphosphate nucleotide to the 3’-ends of single-stranded RNA. Desthiobiotin may be used for detection or as an elutable affinity handle. The length of the spacer between the nucleotide and desthiobiotin has been optimized for efficient attachment to the beads without compromising the accessibility of the RNA to protein.
The procedure for enrichment of RNA binding proteins has been optimized for ease-of-use. The labeled RNA is first captured to the beads to orient the RNA for protein binding. RNA-bound beads are then equilibrated in Protein RNA Binding buffer before protein lysate is added. After washing, sample can be eluted using either nondenaturing Biotin Elution Buffer or SDS-PAGE loading buffer. Eluted samples are ready for a variety of downstream applications, including Western blotting or Mass spectrometry.
|RNA-Protein Pull-Down Protocol. Summary of the procedure for end-labeling RNA with desthiobiotin and then capturing and enriching specific RNA binding proteins using the Thermo Scientific Pierce Magnetic RNA-Protein Pull-Down Kit.
The control system for the pull-down assay utilizes 3'-untranslated region androgen receptor (AR) RNA, poly(A)25 RNA, and mammalian cell lysate. The proximal 3’-untranslated region (UTR) of androgen receptor RNA contains UC-rich regions for HuR and Poly(C) Binding Proteins (CP1 and 2). These RNA binding proteins regulate mRNA stability (HuR) and mRNA turnover and translation (CP1 and 2). The negative control RNA, poly(A)25 RNA, does not contain HuR or poly(C) BP binding sites.
End-labeled RNA enriches specific target binding proteins. RNA binding proteins (RBP) of the AR 3' UTR control system (top panel) and three experimental systems were enriched according to kit procedure. L = lysate; FT = flow-through; E = eluate.
Incubation of A431 lysate with labeled AR UTR RNA (Target) enriches HuR RBP, while incubation with negative control poly(A) RNA (Unrelated) or beads only (None) does not (compare Elution lanes). The same pattern results with the experimental systems, confirming the proper function of the kit. Samples were normalized by volume, and bands were detected using Thermo Scientific SuperSignal West Pico Substrate (Part No. 34080) by a 2-minute exposure to film. Target RNA sequences were as follows:
Androgen Receptor 3’ UTR (Kit Control System):
Tat Exon 2:
|Example RNA-protein pull-down experiment: the effect of mutation on the ability of androgen receptor 3’-UTR RNA to bind HuR and Poly(C)BP. Wild-type and mutant AR 3’-UTR RNA (50pmol) were labeled and used for the pull-down assay according to the kit procedure. Samples were normalized by volume.L = lysate load; FT = flow-through, E = eluate. PCBP1 antibody (1:1000).
- Yeap, B., et al. (2002). Novel binding of HuR and Poly(C)-Binding Protein to a conserved UC-rich motif within the 3’-untranlsated region of the androgen receptor messenger RNA. J. Biol. Chem. 277:27183-92.
- Khanam, T., et al. (2006). Poly(A)-binding protein binds to A-rich sequences via RNA-binding domains 1+2 and 3+4. RNA Biology 3:170-7.
- Piskounova, E., et al. (2008). Determinants of micorRNA processing inhibition by the developmentally regulated RNA-binding protein Lin28. J. Biol. Chem. 283:21310-4.
- Hallay, H., et al. (2006). Biochemical and NMR study on the competition between proteins SC-35, SRp40, and Heterogeneous Nuclear Ribonucleoprotein A1 at the HIV-1 Tat Exon 2 splicing site. J. Biol. Chem. 281:37159-74.
Review of protein-nucleic acid interactions
Methods for detecting protein-RNA interactions
Pierce RNA 3’-End Desthiobiotinylation Kit
Streptavidin Magnetic Beads
Other Nucleic Acid Labeling and Detection Products
Chromatin IP Kit (ChIP)
Active GTPase Pull-Down and Detection Kits
Enzyme Active Site Probes and Enrichment Kits