Rnase A
Ribonuclease A(RNaseA) ndi polypeptide yokhala ndi chingwe chimodzi chokhala ndi ma 4 disulfide bond okhala ndi molekyulu yolemera pafupifupi 13.7 kDa.RNase Ais ndi endoribonuclease yomwe imawononga kwambiri RNA yokhala ndi chingwe chimodzi pa C ndi U zotsalira.Mwachindunji, cleavage imazindikira mgwirizano wa phosphodiester wopangidwa ndi 5'-ribose wa nucleotide ndi gulu la phosphate pa 3'-ribose ya pyrimidine nucleotide yoyandikana, kotero kuti 2,3'-Cyclic phosphates ndi hydrolyzed kuti igwirizane ndi 3'. 'ma phosphates a nucleoside (mwachitsanzo,pG-pG-pC-pA-pG amadulidwa ndi RNase A kuti apange pG-pG-pCp ndi A-PG).RNase A ndiyo yomwe imagwira ntchito kwambiri pakudula RNA ya chingwe chimodzi.Analimbikitsa ntchito ndende ndi 1- 100 μ G/mL, n'zogwirizana ndi machitidwe osiyanasiyana kachitidwe.Mchere wochepa wa mchere (0-100 mM NaCl) ungagwiritsidwe ntchito podula RNA yokhala ndi nsonga imodzi, RNA yamitundu iwiri, ndi RNA unyolo wopangidwa ndi RNA-DNA hybridization.
Komabe, pamchere wambiri (≥0.3 M), RNAse A imangodula RNA yokhala ndi chingwe chimodzi.
RNase A imagwiritsidwa ntchito kwambiri kuchotsa RNA panthawi yokonza DNA ya plasmid kapena genomic DNA.Kaya DNase ikugwira ntchito kapena ayi panthawi yokonzekera ikhoza kukhudza zomwe zimachitika.Njira yachikhalidwe yowira posamba m'madzi ingagwiritsidwe ntchito kuletsa ntchito ya DNase.Mankhwalawa alibe DNase ndi protease, ndipo safuna chithandizo cha kutentha musanagwiritse ntchito.Kuphatikiza apo, mankhwalawa atha kugwiritsidwanso ntchito poyesera mamolekyulu a biology monga kusanthula kwachitetezo cha RNase ndi kusanthula kwa RNA.
Zosungirako
Zogulitsa ziyenera kusungidwa pa -25 ℃ ~ - 15 ℃ kwa zaka 2.
Zofotokozera
| Maonekedwe | Ufa |
| Kuchuluka | 100mg / 1g |
| Mtundu wa mankhwala | Rnase A |
Malangizo
Iyi ndi imodzi mwa njira zodziwika bwino pokonzekera njira yosungirako RNase A.Ikhozanso kukonzedwandi njira zina molingana ndi njira zachikhalidwe mu labotale kapena zolemba zofotokozera (mongaKusungunuka mwachindunji mu 10 mM Tris-HCl, pH 7.5 kapena Tris-NaCl solution)
1. Gwiritsani ntchito 10 mM sodium acetate (pH 5.2) kukonzekera 10 mg/mL ya RNase A yosungirako njira.
2. Kutentha pa 100 ℃ kwa 15 min.
3. Kuzizira mpaka kutentha kwa chipinda, onjezerani 1/10 voliyumu ya 1 M Tris-HCl (pH 7.4), sinthani pH yake kukhala 7.4 (kwaMwachitsanzo, onjezani 500 mL ya 10 mg/mL RNase yosungirako njira 1 M Tris-HCl, pH7.4).
4. Zopakidwa pang'ono -20 ℃ posungirako chisanu, zomwe zimatha kukhazikika kwa zaka ziwiri.
[Zolemba]:
Mukawiritsa yankho la RNaseA mosalowerera ndale, mvula ya RNase idzapanga;Wiritsani pa pH yotsika, ndipo ngati pali mvula, imatha kuwonedwa, yomwe ingayambitsidwe ndi kukhalapo kwa zonyansa zama protein.Ngati matope apezeka atatha kuwira, zonyansa zimatha kuchotsedwa ndi centrifugation yothamanga kwambiri (13000rpm), kenako ndikudzaza kuti muzisungirako kuzizira.
Zolemba
Chonde valani PPE yofunikira, malaya a labu ndi magolovesi, kuti mutsimikizire thanzi lanu ndi chitetezo.
