RiboCluster Profiler™ Ribotrap
Discover the RNA binding protein associated with your gene of interest
Post-transcriptional regulation of gene expression plays important roles in most cellular processes such as development and metabolism and in many diseases such as cancer progression. RNP complexes are the fundamental units of post-transcriptional regulation of mRNA. RNA molecules, including mRNA and non-coding species such as microRNA (miRNA), exist in RNP complexes with specific RNA-binding proteins (RBPs). The RiboCluster Profiler™ RiboTrap Kit utilizes BrU-labeled mRNA to isolate the RBP bound to the gene of interest.
Principle of the RiboCluster Profiler™ RiboTrap
The RiboCluster Profiler™ RiboTrap Kit is used to isolate RBPs and other proteins that are associated with mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA, miRNA or any other RNA of interest from either the cytoplasmic or nuclear extract of cultured mammalian cells. The RNA of interest is modified with 5-bromo-UTP (BrUTP) by in vitro transcription, followed by incubation with a cell lysate (cytoplasmic extract or nuclear extract) to form the assembled RNA-RBP complexes. The BrU-labeled RNA-RBP complexes are then immunoaffinity purified by anti-BrdU monoclonal antibody (mAb), which cross-reacts with BrUTP. RBPs associated with the BrU-labeled RNA can then be identified by immunoblotting or mass spectroscopy.
Three different wash buffers provided in the RiboCluster Profiler™ RiboTrap Kit allow for analysis of both weakly and tightly bound RBPs. The wash buffer with mild conditions (Wash Buffer I) is used for primary screening of total RBPs, while wash buffer with stringent conditions (Wash Buffer II or III) is suitable for purifying tightly bound RBPs. Antisense RNA (corresponding to complimentary RNA of interest) or truncated RNA of interest is used to exclude nonspecific binding proteins. Elution buffer composed of the optimal concentration of BrdU allows specific recovery of BrU-RNA/protein complexes. RBPs isolated with native conformations can be used in several downstream applications.
- Immunoaffinity method to explore RNA proteins and RNA-RNA interactions
- Reliable method to describe the endogenous assembly of ribonucleoproteins
- Identify regulatory components in biosynthesis of small RNA and non-coding RNA
Advantage of using RiboCluster Profiler™ RiboTrap
- RNA of interest can be analyzed using cytoplasm or nuclear fraction
Example of RiboCluster Profiler™ RiboTrap Experiment
BrU-labeled 3'UTR of p21 mRNA was incubated with cytoplasmic fraction of HEK293T cells to form mRNA-protein complex followed by immunoaffinity purification using anti-BrdU antibody. Bands ①, ② and ③ are identified to be IGF2BP1/IMP1, PCBP2 and HuR by LC-MS/MS