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  • p19 siRNA Binding Protein

    Description

    The p19 siRNA Binding Protein (19 kDa) from the Carnation Italian Ringspot Virus (CIRV) plant binds siRNAs with nanomolar affinity(1). The dimeric protein binds 21 base siRNAs with a 2 base 3´ extension and a 5´phosphate. The protein binds RNA in a size dependent and sequence independent manner. If the siRNAs are 4 bases longer, the affinity for the protein is reduced about 100-fold (2). When p19 siRNA Binding Protein is expressed in plants it suppresses RNA interference (3).

    Figure 1:
    Size specific binding of siRNA by p19 siRNA Binding Protein. Three phosphorylated dsRNAs of 17,21 and 25 bases (30 ng each band) were mixed with increasing amount of p19 siRNA Binding Protein (0-3 µg) in a 20 µl reaction, and incubated at room temperature for 1 hour. The reaction was analyzed on a 20% polyacrylamide gel stained with ethidium bromide. Marker M is the siRNA Marker (NEB #N2101 ).

    Highlights

    • For the isolation of siRNA
    • Isolated from a recombinant source

    Product Source

    p19 siRNA Binding Protein is cloned and expressed in E. coli as a fusion protein with an amino terminal MBP (maltose binding protein) and a carboxy terminal CBD (chitin binding domain).

    Advantages and Features

    Applications

    High affinity binding of siRNAs
    Affinity purification of siRNA with chitin magnetic beads

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of protein that binds to 10 ng of siRNA at 25°C in 1 hour.

    Unit Assay Conditions
    1 unit of p19 siRNA Binding Protein was incubated with 20 ng of phosphorylated siRNA in 1X p19 siRNA Binding Buffer in a volume of 20 µl at 25°C for 1 hour.

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    150 mM NaCl
    0.5 mM DTT
    1 mM EDTA
    50% Glycerol
    pH 7.5 @ 25°C

    Quality Control

    Quality Assurance Statement

    • p19 siRNA Binding Protein contains no detectable DNases, RNases and phosphatases. The purified protein contains no detectable DNA or RNA as determined by ethidium bromide staining of an agarose gel.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

    Notes

    1. p19 siRNA Binding Protein can selectively bind siRNAs in the presence of a 2,000 fold excess of other RNAs.
    2. 1X p19 siRNA Binding Buffer:
      20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM TCEP, 0.02% Tween-20 (pH 7.0 @ 25°C)
    3. p19 siRNA Binding Protein does not bind ssRNA.Double-stranded 21 bp RNA with a 2-base overhang and a 5´ phosphate binds the most efficiently. Double-stranded miRNAs with mismatched base pairs will bind with lower affinity.
    4. Due to the difference in molecular weight between p19 siRNA Binding Protein and siRNA, a 10 to 20-fold excess of p19 siRNA Binding Protein is needed.
    5. TCEP (Tris 2-carboxyethyl Phosphine) can be replaced with DTT (dithiotreitol) in the required solutions. However, if DTT is used, it needs to be added separately into the reaction.

    References

    1. Silhavy, D. et al. (2002). EMBO J. 21
    2. Vargason, J.M., et al. (2003). Cell. 115
    3. Qiu, W. et al. (2002). Mol. Plant Microbe Interact. 15

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Protocol for isolation of siRNA from p19 siRNA Binding Protein (M0310) using chitin magnetic beads

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