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RBP (RNA Binding Protein) / RIP-Assay Kit

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  1. What type of experimental procedure is RIP?
    RNP* immunoprecipitation (RIP) is a procedure used in studying RNAs binding to RBPs (RNA Binding Protein) by isolating the RNPs through immunoprecipitation using specific antibodies against RBPs.

    *RNP is a complex formed between RNA and RBP in a cell.
  2. What is peculiar about the samples obtained from the RIP procedure?
    It is expected that the" RNAs directly binding RBPs" and "RNAs indirectly binding RBPs such as RNA interacting with other RBP formed complex with target RBP" in the sample become concentrated. When comparing the rRNA and the tRNA in the sample after RIP with mRNA (and miRNA) which was initially just a small percentage of the total RNA, we find out that the concentration has increased. In addition, analysis for the enriched RNAs are low background and able to easily find out their relationship (for example, working at same pathway).
  3. What are the different methods used in analyzing the samples obtained for the RIP procedure?
    ・Measuring the quantity of the UV (A260nm) absorbed (MBL uses NanoDrop)
      However, this method cannot be used on low concentration samples due to low sensitivity.

    ・Waveform analysis using an Agilent Bioanalyzer
    → It is possible to test with high sensitivity the size distribution of the nucleic acids in a RIP sample (capillary electrophoresis).
    ・Sequence determination using sequencing (classical sequencing)
    → The base sequence of the RBPs can be determined.
    ・Real-time quantitative PCR analysis
    → Specific detection and semi-quantitative analysis.
    ・Microarray Analysis
    → A filter is available to narrow down analysis results from tens of thousands of mRNA.
    ・High-throughput sequencing
    → The base sequence of the RBPs can be determined.
  4. Can it be used when performing RIP-Assay with in-house antibodies?
    The ability of RIP differs depending on the antibodies used. At times, RNA is not isolated even upon precipitation of RBPs. Hence, we recommend the use of purified antibodies and an examination of whether sufficient quantity/quality of RNA is isolated and if the addition of High-Salt Solution is necessary before performing the actual experiment.
  5. Can you carry out RIP-Assay using Tag antibodies?
    We have confirmed that anti-Myc-tag antibody (code. M047-3(clone: PL14)) from MBL can be used in RIP. Other antibodies have not been tested.
  6. Can you isolate the RNP clusters in the nucleus?
    The RIP-Assay Kit is intended for analyzing the "RNP in the cytoplasm". Lysis buffer provided RIP-Assay Kit does not completely solublize nuclear membrane, hence nuclear RNPs can be hardly collected.
  7. About the fact that the RIP-Assay Kit is unsuitable for nuclear RNA isolation, are there any ways to resolve this?
    If you do not perform RIP-Assay without cross-linking, using RiboTrap Kit and RIP-Assay Kit for microRNA will enable RIP-Assay for nuclear RNPs. Please prepare nuclear lysate according to the procedure of RiboTrap Kit, and then perform RIP-Assay. In this case, it's better to optimize sonic condition to reduce DNA contamination without RNA degradation.
  8. How many cells are needed for RIP-Assay?
    It differs depending on the cell but please prepare a sample of 4×106-2×107 cells. Initially, we recommend considering a 1×107cells/sample.
  9. I would like to use magnetic beads. Is it possible?
    Dynabeads® (Invitrogen) can be used for Protein A and Protein G. You can also use Protein G-Magnetic Beads (MBL; code. MJS002V2).
  10. Is there any particular recommendation or warning when using beads that can be used with the RIP-Assay Kit?
    In some case, the background may be increased depending on how hard the cross-linking is when using agarose beads.
    We recommend reagents such as the listed below;
    Protein A (or G) Sepharose CL-4B (GE Healthcare)
    Immobilized Protein G Plus (Pierce; code. 22852)
  11. I would like to do RIP-Assay with cross-linking using RIP-Assay Kit. Is it possible?
    RIP-Assay Kit does not support cross-linking. You will need some protocol modifications.
  12. Can the RNA be stabilized just by adding Protease inhibitors without carrying out cross-linking?
    The addition of protease inhibitors is effective for preventing degradation of the antibodies and the RBPs rather than stabilizing interaction between RNAs and RBPs. You will need cross-linking if you want to collect RNAs weakly bound to RBPs.
  13. Is RIP-Certified antibody useful for RIP-Assay with (formalin) cross-linked?
    We have not examined cross-linking in formalin.
    In formalin, not only the nucleic acid and protein, but also between proteins are cross-linked.
    Therefore, we think formalin is not very suitable for RIP-Assay after cross-linking.
  14. What is the difference between RIP-Assay Kit and RIP-Assay Kit for microRNA?
    RIP-Assay Kit cannot collect small RNAs. RIP-Assay Kit for microRNA can collect both small and large RNAs. Furthermore, it can also collect small and large RNAs separately as well as collect only small RNAs. By collecting small RNAs bound to RBPs/large RNAs, you will able to study how your interesting intracellular event is regulated by not only RBPs but also small RNAs. In addition, it is also useful for the identification of target RNAs specific binding to your interested miRNAs.
  15. When amplifying the target sequence by RT-PCR, how much RNA is required for reverse transcription after its purification?
    To compare the target RNA with negative control, you will use equal amounts of RNAs. To compare the target RNA with total RNA (RIP RNA vs toal RNA), you will use equal volume of RNAs. Please see the following example:
    If you have the following three as a sample and use 1 ng as a template for RT-PCR:
    1. Control IgG, (concentration: 2 ng/μL)
    2. RBP-RIP RNA, (concentration: 20 ng/μL)
    3. Total RNA, (concentration: 100 ng/μL)
    You will use each 1 ng of sample 2 and sample 3. To compare sample 1 with sample 2, you will use 1/20 μL of sample 2 and 1/20 μL of sample 1.
  16. I would like to compare RIP RNA with negative control in microarray. Is it possible?
    In microarray, most of RNA expression patterns do not change but a part of them drastically changes. Hence it makes possible to normalize and analyze array data. However, in RIP-Chip, the comparison target RBP-RIP with negative control or other RBP-RIP is nonsense bacause the RNA population is mostly different from each other. Microarray is good for comparison of different two conditions such as drug-treated or not, and gene transfectant or mock.
  17. Can the RIP-Assay Kit be used for the liver tissue sample?
    Yes. It can be used for the liver tissue sample.