Rnase Inhibitor
Murine RNase inhibitor yog recombinant murine RNase inhibitor qhia thiab purified los ntawm E.coli.Nws khi rau RNase A, B lossis C hauv qhov sib piv 1: 1 los ntawm kev sib txuas tsis yog covalent, yog li inhibiting kev ua haujlwm ntawm peb enzymes thiab tiv thaiv RNA los ntawm degradation.Txawm li cas los xij, Nws tsis zoo rau RNase 1, RNase T1, S1 Nuclease, RNase H lossis RNase los ntawm Aspergillus.Murine RNase inhibitor tau kuaj los ntawm RT-PCR, RT-qPCR thiab IVT mRNA, thiab tau sib xws nrog ntau yam kev lag luam rov qab transcriptases, DNA polymerases thiab RNA polymerases.
Piv rau tib neeg RNase inhibitors, murine RNase inhibitor tsis muaj ob lub cysteine rhiab heev rau oxidation uas ua rau inactivation ntawm inhibitor.Qhov ntawd ua rau nws ruaj khov ntawm qhov tsis tshua muaj siab ntawm DTT (tsawg dua 1 mM).Cov yam ntxwv no ua rau nws tsim nyog rau kev siv cov tshuaj tiv thaiv uas muaj qhov siab ntawm DTT tsis zoo rau cov tshuaj tiv thaiv (xws li Real-time RT-PCR).
Adaim ntawv thov
Cov khoom no tuaj yeem siv dav hauv txhua qhov kev sim uas RNase cuam tshuam tau kom tsis txhob RNA degradation, xws li:
1.Synthesis ntawm thawj strand ntawm cDNA, RT-PCR, RT-qPCR, thiab lwm yam.
2.Tiv thaiv RNA los ntawm degradation thaum lub sij hawm hauv vitro transcription / txhais lus (xws li, hauv vitro viral replication system).
3.Inhibition ntawm RNase kev ua si thaum lub sij hawm RNA sib cais thiab purification.
Kev cia khoom
Cov khoom tuaj yeem khaws cia ntawm -25 ~ 15 ℃, siv tau rau 2 xyoos.
Cia tsis
50 mM KCl, 20 mM HEPES-KOH (pH 7.6, 25 ℃), 8 mM DTT thiab 50% glycerol.
Unit txhais
Tus nqi ntawm murine RNase inhibitor yuav tsum tau inhibit qhov kev ua ntawm 5ng ntawm ribonuclease A los ntawm 50% tau txhais tias yog ib chav tsev (U).
Molecular hnyav ntawm cov protein
Qhov hnyav molecular ntawm murine RNase inhibitor yog 50 kDa.
Kev tswj kom zoo
Exonuclease Kev ua:
40 U ntawm murine RNase inhibitor nrog 1 μg λ -Hind III digest DNA ntawm 37 ℃ rau 16 teev yields tsis degradation raws li txiav txim los ntawm agarose gel electrophoresis.
Kev Ua Haujlwm Endonuclease:
40 U ntawm murine RNase inhibitor nrog 1μ g λ DNA ntawm 37 ℃ rau 16 teev yields tsis degradation raws li kev txiav txim los ntawm agarose gel electrophoresis.
Nicking Kev ua:
40U ntawm murine RNase inhibitor nrog 1μ g pBR322 ntawm 37 ℃ rau 16 teev yields tsis degradation raws li txiav txim los ntawm agarose gel electrophoresis.
RNase Kev ua si:
40U ntawm murine RNase inhibitor nrog 1.6μ g MS2 RNA rau 4h ntawm 37 ℃ yields tsis degradation raws li txiav txim los ntawm agarose gel electrophoresis.
E.coli DNA:
40 U ntawm murine RNase inhibitor yog tshuaj xyuas qhov muaj E. coli genomic DNA siv TaqMan qPCR nrog primers tshwj xeeb rau E. coli 16S rRNA locus.E. coli genomic DNA paug yog ≤ 0.1 pg / 40 U.
Nua otes
1.Vim li cas oscillation los yog nplawm yuav ua rau enzyme inactivation.
2.Qhov zoo tshaj qhov kub ntawm qhov no tus inhibitor yog 25-55 ℃, thiab nws yog inactivated ntawm 65 ℃ thiab saum toj no.
3.Cov kev ua ub no ntawm RNase H, RNase 1 thiab RNase T1 tsis tau inhibited los ntawm murine RNase inhibitor.
4.Qhov inhibition ntawm RNase kev ua haujlwm tau pom nyob rau hauv ntau yam ntawm pH (pH 5-9 yog tag nrho cov nquag), thiab kev ua haujlwm siab tshaj plaws tau pom ntawm pH 7-8.
5.Txij li thaum ribonucleases feem ntau khaws cov kev ua haujlwm nyob rau hauv cov xwm txheej denaturing, yuav tsum tau saib xyuas kom tsis txhob muaj cov RNase Inhibitor molecules uas muaj complex nrog ribonuclease.Txhawm rau tiv thaiv kev tso tawm ntawm cov nquag ribonuclease, qhov kub siab tshaj 50 ° C thiab siab concentrations ntawm urea los yog lwm yam denaturing agents yuav tsum raug zam.
