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RiboTrap Kit

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  1. What is RiboTrap?
    RiboTrap is an immunoaffinity method to isolate RNA-binding proteins by using RNA of your interest as bait. This method enables to isolate unknown proteins as well as known proteins.
  2. What is the difference between RiboTrap and EMSA (gel shift assay)?
    RiboTrap is used to identify proteins that bind to a specific RNA. It enables to identify unknown proteins as well as known proteins. EMSA is used to detect the binding between specific sequences of nucleic acids and proteins.
  3. How do you perform BrU-RNA synthesis?
    We synthesize BrU-labeled RNA by adding BrUTP to the reactive substrate of in vitro transcription. The recommended molar ratio of BrUTP to standard UTP is 1:1 to 1:3. To determine the appropriate ratio, uracil content in the RNA sequence should be considered.
  4. How much are BrUTPs incorporated into the RNA?
    The incorporation rate of BrUTP depends on the uracil content of the RNA sequence and BrUTP-adding ratio. Moreover, BrUTPs are randomly incorporated into the RNA when performing in vitro transcription. We recommend using synthesized oligonucleotides as bait if you need BrU labeling at the specific positions in the RNA.
  5. Is there any possibility that BrU labeling inhibits the binding of RNAs and proteins?
    According to our in-company data, we think that the possibility is low.
  6. Is it possible to isolate proteins binding to small ncRNA using RiboTrap?
    We conclude that small ncRNAs can be used for RiboTrap. The BrU-labeled positions and numbers in the target sequence should be considered when performing RiboTrap using short-length RNAs. Synthesized RNA oligos can be used as bait if necessary.
  7. Is there any possibility that unincorporated BrUTPs after in vitro transcription inhibit the binding between antibody and BrU-RNA?
    Unincorporated BrUTPs inhibit the interaction between BrU-RNAs and antibodies via competitive binding. Thus, synthesized BrU-RNA should be purified after in vitro transcription.
  8. Do you confirm the incorporation of BrU into synthesized RNA after in vitro transcritption?
    We confirm the incorporation of BrU as follows:
  9. How much protein should be used in RiboTrap Kit?
    We have not adjust the amount of protein, but we start experiments with 7-10x107 cells.
  10. What is the difference among three Wash Buffers?
    ・Wash Buffer I: The buffer with the mildest conditions. It allows the isolation of weakly-bound proteins as well as tightly-bound proteins. On the other hand, the mild condition gives higher background than Wash Buffer II and III.
    ・Wash Buffer II: The buffer with high ionic strength. It is suitable to isolate the proteins that tightly bind to RNAs.
    ・Wash Buffer III: The buffer with high detergency. It reduces proteins that indirectly bind to the target RNA.
    Please select the wash buffer best suited for your purpose.
  11. Have you added tRNA to reduce nonspecific binding?
    We have not tried to use tRNA as blocking. Nonspecific binding can be avoided by washing with Wash Buffer included in RiboTrap Kit. For optimization of washing condition, we recommend using Wash Buffer I for primary screening and then using Wash Buffer II or III if needed.
  12. Is there any possibility that RNAs are degraded when mixing BrU-labeled RNAs and Cell Lysate?
    RNase inhibitor is added in the Lysis Buffer. In RiboTrap as well as RIP-assays, please take all possible measures to avoid RNA degradation such as using nuclease-free grade reagents and instruments, wearing masks and gloves, and controlling temperature of the experiment environment.
  13. Are there any in vitro transcription kits that you recommend?
    We recommend following in vitro transcription kits;
    ・CUGA®7 in vitro Transcription Kit (Nippon Gene; code. 307-13531)
    ・Riboprobe® System-T7 (Promega; code. P1440)
    ・TranscriptAidTM T7 High Yield Transcrition Kit (Thermo Fisher Scientific; code. K0441)
  14. Are there any recommended beads suitable for RiboTrap Kit?
    We recommend Immobilized Protein G Plus (Thermo Fisher Scientific; code. 22852). Dynabeads® (Invitrogen; code. 10003D) and Protein G-Magnetic Beads (MBL; code. MJS002V2) also can be used in RiboTrap experiments.
  15. Is it possible to isolate proteins binding to long-length RNA such as Xist using RiboTrap?
    We have not tried to isolate proteins binding to lncRNA with RiboTrap. However, it is possible by using several truncated forms of the RNA as bait.
  16. Is it possible to isolate miRNAs as well as proteins using RiboTrap Kit?
    The isolation of miRNA is also possible. After the formation of antibody-bait RNA-RNP complex and washing steps, we have isolated miRNAs by Separation method of RNA extraction protocol in RIP-Assay Kit for microRNA. The isolated miRNAs were then cloned and sequenced. Depending on the RNA of your interest, amounts of isolated miRNAs may be small compared to the RIP-Assay with antibodies against RISC components. Therefore, we recommend scaling up the experiment.