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 A 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 ku 3 yofanana. ′ nucleoside phosphates (mwachitsanzo pG-pG-pC-pA-pG imadulidwa ndi RNase A kupanga pG-pG-pCp ndi A-PG).RNase A ndiyo yomwe imagwira ntchito kwambiri pakudula RNA ya chingwe chimodzi.Kukhazikika kovomerezeka kogwirira ntchito ndi 1-100μG/mL, kumagwirizana ndi machitidwe osiyanasiyana.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 pochotsa RNA panthawi yokonzekera DNA ya plasmid kapena genomic DNA.Kaya kapena DNase ikugwira ntchito 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 komanso kusanthula kwa ma RNA.
Zosungirako
Mankhwala akhoza kusungidwa pa -25～-15 ℃, zomveka 2 zaka.
Malangizo
Iyi ndi imodzi mwa njira zodziwika bwino pokonzekera njira yosungirako RNase A.Itha kukonzedwanso ndinjira 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) pokonzekera 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 10g/ml RNase yosungirako njira 1M Tris-HCL, PH7.4)
4. Yoyikidwa pa -20 ℃ posungirako chisanu, yomwe imatha kukhazikika kwa zaka ziwiri.
[Zidziwitso]: Mukawiritsa yankho la RNaseA mosalowerera ndale, mvula ya RNase ipanga;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 mozizira.
Zambiri zamalonda
| Mawu ofanana ndi mawu | Ribonuclease I;Pancreatic ribonuclease;Ribonuclease 3'-pyrimidnooligonucleotidohydrolase;Rnase A;Endoribonulcease I |
| CAS No. | 9001-99-4 |
| Maonekedwe | White lyophilized ufa |
| Kulemera kwa maselo | ~ 13.7kDa (kutsatizana kwa amino acid) |
| Ph Mtengo | 7.6 (ntchito 6-10) |
| Kutentha Koyenera | 60 ℃ (Zochita zosiyanasiyana 15-70 ℃) |
| Wothandizira | Na2+.K+ |
| Choletsa | Rnase Inhibitor |
| Inactivation njira | Sangathe kuyambitsidwa ndi kutentha, tikulimbikitsidwa kugwiritsa ntchito centrifuge column |
| Chiyambi | Ng'ombe |
| Kusungunuka | Zosungunuka m'madzi (10mg/ml) |
| Kutayika pouma | ≤5.0% |
| Ntchito ya enzyme | ≥60 Kunitz mayunitsi/mg |
| Isoelectric point | 9.6 |
Zolemba
Pa chitetezo ndi thanzi lanu, chonde valani malaya a labu ndi magolovesi otayika kuti mugwire ntchito.
