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Protocols for p19 siRNA Binding Protein FAQs for RNAi and RNA Enzymes Technical Reference for RNAi and RNA Enzymes Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE 12-Tube Magnetic Separation Rack 6-Tube Magnetic Separation Rack Anti-CBD Monoclonal Antibody Anti-MBP Monoclonal Antibody Chitin Beads Chitin Magnetic Beads

Home > Products > RNAi and RNA Enzymes > RNA Isolation and Analysis > p19 siRNA Binding Protein p19 siRNA Binding Protein Catalog # Size Concentration Price Qty   M0310L 5,000 units 10,000 units/ml $272.00 M0310S 1,000 units 10,000 units/ml $68.00 Prices are in US dollars and valid only for US orders. Download: MSDS PDF For the isolation of siRNA Isolated from a recombinant source 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). 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). Applications: High affinity binding of siRNAs Affinity purification of siRNA with chitin magnetic beads Reagents Supplied: p19 siRNA Binding Buffer (10X) Reaction & Storage Conditions Incubate at 25°C. 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. Concentration: 10,000 units/ml Storage Conditions: 10 mM Tris-HCl 150 mM NaCl 0.5 mM DTT 1 mM EDTA 50% Glycerol pH 7.5 @ 25°C Storage Temperature: -20°C Notes General notes: 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) Usage notes: p19 siRNA Binding Protein can selectively bind siRNAs in the presence of a 2,000 fold excess of other RNAs. 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. 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. 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. Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product. 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. Exonuclease Activity: Incubation of a 50 μl reaction containing 100 units of p19 siRNA Binding Protein with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC released < 0.1% of the total radioactivity. Endonuclease Activity: Incubation of a 50 μl reaction containing 100 units of p19 siRNA Binding Protein with 1 μg of ΦX174 RF I DNA for 4 hours at 37ºC resulted in < 5% conversion to RFII as determined by agarose gel electrophoresis. Nuclease Activity: Incubation of 100 units of p19 siRNA Binding Protein for 16 hours at 37°C in 50 µl of p19 siRNA Binding Buffer with 1 µg λ DNA yielded a clear and sharp band on an agarose gel. References Silhavy, D. et al. (2002) EMBO J., 21, 3070-3080. Vargason, J.M., et al. (2003) Cell, 115, 799-811. Qiu, W. et al. (2002) Mol. Plant Microbe Interact, 15, 269-280. Companion Products 12-Tube Magnetic Separation Rack 6-Tube Magnetic Separation Rack Anti-CBD Monoclonal Antibody Anti-MBP Monoclonal Antibody Chitin Beads Chitin Magnetic Beads