RNase inhibitors

The crystal structure of one LRR protein, the RNAse inhibitor, has revealed that leucine-rich repeats correspond to p-a structural units. This units are arranged for a parallel p-sheet with one surface exposed to solvent so that the protein acquires an unusual non-globular shape, which may be responsible for proteinbinding functions [57]. 

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

The collected polysomal fraction also contains a significant amount ofRNases. An overload of RNases cannot be completed by the commonly used RNase Inhibitors (rRNasin, RNase Inhibitor, etc.) and will therefore lead to massive degradation. It is therefore recommended not to collect more than the amount indicated here. 

Most often, the ISH phase is done last because some antigens may be destroyed by the high temperature and protein digestion of ISH. Conversely, the antibodies used for immunocytochemistry may have some RNase carryover that will diminish transcript targets. The addition of RNase inhibitors, therefore, may be warranted (12). 

Select slides with optimized proteinase K digestion and follow the steps below for DNase digestion. Prepare in an Eppendorf tube 1.0 pL RNasin (RNase inhibitor), 11.5 pL UV-irradiated dH20, 37.5 U DNase (RQl RNase free DNase, Promega Inc., Madison, Wl)/(1 U/pL) Total (per slide) 50.0 pL. 

DTT-dependent and -independent RNAse inhibitors (Ambion), NTPs, dNTPs, MgClj, MnCl, yeast t-RNA, DNA, and RNA molecular weight standards. 

The addition of RNase inhibitor (50 units) to the translation reaction mixture (50 pL) may improve translational efficiency (Recommended products Promega Code No. N2611). 

The addition of RNase inhibitor is to preserve the full-length smgle-stranded RNA transcripts... 

All previous and subsequent steps of the RNA selection process should be performed following sPict RNase-free conditions. This includes the use of DEPC-Peated H20 for all solutions and the use of aerosol barrier pipette tips. Any glassware used (including elecPophoresis and elecPoelution apparatus) should be Peated with an RNase inhibitor spray (e. g., RNase AWAY, Molecular BioProducts). 

Murphy, N.R., S.S. Leinbach, and R.J. Hellwig. 1995. A potent, cost-effective RNase inhibitor. Biotechniques 18 1068. 

SP6 RNA polymerase and RNase inhibitor (Takara, Kyoto, Japan or Promega, Madison, WI). 

RNasin Plus RNase inhibitor, Promega (N2615)... 

RNase H buffer, 12.5 fil RNasin Plus RNase inhibitor, and 50 /(I RNase H enzyme to 137.5 fil nuclease-free H20. Add this to the annealing reaction. Incubate at 37 °C for 2 h. Stop the reaction with l/20th volume 0.5 M EDTA, pH 8.0. 

The E. coli ribosome display system had to be considerably optimized for efficient display of folded proteins. These improvements (explained in more detail in Section III, B) involved the use of RNase inhibitors, the design of hairpins at either end of the RNA and a separate transcription and translation step allowing control over the individual redox requirements, and lead to higher yields, greater stability, and reduced nonspecific binding of the complexes (Hanes and Pliickthun, 1997 Hanes et al., 1999). 

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