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.
Features of the Magnetic RNA-Protein Pull-Down Kit:
• 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
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).
Includes: 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
Requires: User-supplied RNA and lysate (experimental sample)
Applications: • 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.
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.
Sufficient For: 20 RNA-protein complex pull-down reactions • Pierce Nucleic Acid-Compatible Streptavidin Magnetic Beads, 1 mL (store at 4°C) • RNA Capture Buffer (1X), 10 mL (store at 4°C) • Tris (20 mM, pH 7.5), 5 mL (store at 4°C) • Protein-RNA Binding Buffer (10X), 1 mL (store at 4°C) • Wash Buffer (1X), 10 mL (store at 4°C) • Biotin Elution Buffer, 1.5 mL (store at 4°C) • HuR Monoclonal Antibody (Mouse), 50 µL (store at 4°C) • Positive RNA Control (AR RNA), 250 pmol (store at -20°C) • Negative RNA Control (polyA25 RNA), 250 pmol (store at -20°C) • Pierce RNA 3' End Desthiobiotinylation Kit (part number 20163; store at -20°C)