Cell Signaling RNasin® Plus RNase Inhibitor Product RNasin® Plus RNase Inhibitor For Laboratory Use. Description: RNasin® Plus RNase Inhibitor is a recombinant mammalian RNase inhibitor that is expressed as a soluble protein in E. coli, allowing easy purification through a combination of ion exchange and hydrophobic interaction chromatography. The protein is capable of inhibiting eukaryotic RNases (e.g., RNase A and RNase B) similarly to human placental RNase inhibitor. RNasin® Plus RNase Inhibitor is tested in RT-PCR and is compatible with enzymes such as AMV, M-MLV and ImProm-II™ Reverse Transcriptases or Taq and Tfl DNA Polymerases. RNasin® Plus RNase Inhibitor also is tested and compatible with quantitative, real-time RT-PCR in a TaqMan® assay. The inhibitor offers increased resistance to oxidation over the human version of the protein. Two cysteines in the human protein have been identified as especially sensitive to oxidation and react by forming a disulfide bond that can block the active site of the inhibitor. RNasin® Plus, through natural amino acid diversity, lacks the ability to form this site-blocking disulfide. In addition, the new protein has characteristics never before realized, including continued inhibition of RNases above 50°C. Heating solutions of RNasin® Plus and RNase followed by cooling does not result in the reappearance of RNase activity— even when the solution is heated above the denaturation temperature of the RNasin® Plus protein alone. This allows RNasin® Plus to protect RNA species prior to, during and after heating, even at temperatures normally used during first-strand DNA synthesis in RT-PCR. We have taken solutions up to 70°C for 15 minutes and did not see RNase reactivation. Features: • Improved Resistance to Oxidation: Due to natural amino acid diversity, RNasin® Plus lacks the capability to form the active site-blocking disulfide bond that can form in the human protein under oxidative conditions. • Improved Purification: RNasin® Plus is expressed by E. coli as a soluble protein, allowing easy purification by a combination of ion exchange and hydrophobic interaction chromatography. No direct affinity chromatography required. The new process yields a >90% pure protein with no E. coli RNase carryover. • Proven Compatibility with RT-PCR Systems: RNasin® Plus has proven compatible with the Access and AccessQuick™ RT-PCR Systems, ImPromII™ Reverse Transcription System and the Reverse Transcription System. Also proven compatible with TaqMan®-based RT-PCR Systems. • Protection During RNA Template Denaturation: Heating mixtures of RNasin® Plus and RNase does not lead to reactivation of the RNase at temperatures even as high as 70°C for 15 minutes. Many RT-PCR protocols call for RNA template denaturation (e.g., 65–70°C for 5–10 minutes) in the presence of the RT primers prior to full RT reaction assembly for maximum sensitivity. You can now include RNasin® Plus at this step. • Protection During Higher Temperature RT Reactions: Add RNasin® Plus during RT reaction assembly and take the reaction to temperatures above 50°C with enzymes like the ImProm-II™ and AMV Reverse Transcriptases. RNases that may be present will not be reactivated at the higher temperature. • Choose Your Configuration: Learn more about our custom options for this product at: www.promega.com/myway/ Storage Conditions: Store at –20°C. Protocol RNasin® Plus RNase Inhibitor Product Information Part# 9PIN261 Size Conc. Cat.# Price (Fr) 2,500 u 40 u/µl N2611 186.00 10,000 u 40 u/µl N2615 465.00 5 Protection from RNase at 70°C. Separate tubes of RNasin® Plus and RNase (lanes 1 and 3) were heated to 70°C for 15 minutes. RNasin® Plus and RNase were combined and then heated to 70°C for 15 minutes (lanes 2 and 4). To each set of reactions, either 100ng (lanes 1 and 2) or 10ng (lanes 3 and 4) of Luciferase Control RNA (Cat.# L4561) were added. The reactions were held at 37°C for 1 hour, then used in an RT-PCR to amplify the entire 1.8kb transcript. The gel shows the amplified product from the RT-PCR. All lanes used 400u of RNasin® Plus and 1.25μg of a rat liver protein extract (abundant source of RNase; Sigma Cat.# L-1380) dissolved in water to 0.5μg/μl. 12 34 For complete and up-to-date product information visit: www.promega.com/catalog 129 DNA and RNA Purification 4416TB12_3B Pagina 132
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