Monday, 5 December 2011

RiboTrap: Global Analysis of RNP clusters using RNA affinity tags


The RiboTrap Kit  from MBL 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. 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 RiboTrap Kit utilizes BrU-labeled mRNA to isolate the RBP bound to the gene of interest. 

Principle of the RiboTrap Kit

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 RiboTrap Kit allow for analysis of both weakly and tightly bound RBPs. The wash buffer with mild conditions (Wash Buffer I) is usually 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 complementary 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.


For more information on RiboTrap click here.

1 comment:

  1. Thanks a lot for this contribution! It's been very useful for me. Everything is very open and represents very clear explanation of issues. Really blogging is spreading its wings quickly. Your write up is a good example of it. Your website is very useful. Thanks for sharing.
    RNA Purification Kits

    ReplyDelete

Webmaster: AnnWebCom | Site design: custardcreative.co.ukCustard Creative Copyright 2005-2015 Caltag-Medsystems Limited - all rights reserved