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Home › Products › p19 siRNA Binding Protein p19 siRNA Binding Protein Catalog # M0310S Size 1,000 units Concentration 10,000 units/ml Price $78.00 Qty M0310L 5,000 units 10,000 units/ml $312.00 1 1 Categories: miRNA & siRNA Products, RNA Isolation & Detection Products Applications: RNA Detection & Isolation Product Information Protocols & Manuals Other Tools & Resources Description Properties and Usage Notes Quality & Safety Legal Information Advantages and Features Related Products References 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). Reagents Supplied The following reagents are supplied with this product: Store at (°C) p19 siRNA Binding Buffer (200 μl) Concentration 2X 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 Related Products Companion Products 12-Tube Magnetic Separation Rack 6-Tube Magnetic Separation Rack Anti-CBD Monoclonal Antibody Anti-MBP Monoclonal Antibody Chitin Resin Chitin Magnetic Beads 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 Protocols Protocols Datacards 1. Protocol for isolation of siRNA from p19 siRNA Binding Protein (M0310) using chitin magnetic beads 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 [email protected] or fill out the Technical Support Form for appropriate document. M0310Datasheet-Lot0031205 M0310Datasheet-Lot0031402 M0310Datasheet-Lot0031405 M0310Datasheet-Lot0031410 M0310Datasheet-Lot0031501 Selection Charts Interactive Tools Selection Charts Reagents for RNA Library Preparation Interactive Tools NEBioCalculator Quality Control Datacards Safety Data Sheet 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. Further information regarding NEB product quality can be found here. 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. Safety Data Sheet The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely. p19 siRNA Binding Protein p19 siRNA Binding Buffer (200 μl) 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 [email protected] or fill out the Technical Support Form for appropriate document. M0310Datasheet-Lot0031205 M0310Datasheet-Lot0031402 M0310Datasheet-Lot0031405 M0310Datasheet-Lot0031410 M0310Datasheet-Lot0031501 Legal and Disclaimers Legal and Disclaimers This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications. For more information about commercial rights, please contact NEB's Global Business Development team at [email protected]. This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals. Research Use Only The buyer/user has a non-exclusive license to use the vector for Research Purposes Only. Commercial use of this vector requires a license from New England Biolabs, Inc. U.S. Patent No. 5,643,758 Appln. No. WO2007/120809 Trademarks NEW ENGLAND BIOLABS® is a registered trademark of New England Biolabs, Inc. Disclaimers This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.