Rnase A
Ribonuclease A (RNaseA) jẹ polypeptide kan-okun kan ti o ni awọn asopọ disulfide 4 pẹlu iwuwo molikula ti o to 13.7 kDa.RNase A jẹ ẹya endoribonuclease ti o ni pataki degrades nikan-stranded RNA ni C ati U iṣẹku.Ni pato, cleavage mọ asopọ phosphodiester ti a ṣe nipasẹ 5'-ribose ti nucleotide ati ẹgbẹ fosifeti lori 3'-ribose ti pyrimidine nucleotide ti o wa nitosi, ki awọn 2, 3'-Cyclic phosphates ti wa ni hydrolyzed si 3 ti o baamu. phosphates nucleoside (fun apẹẹrẹ pG-pG-pC-pA-pG ti pin nipasẹ RNase A lati ṣe ipilẹṣẹ pG-pG-pCp ati A-PG).RNase A jẹ ohun ti o ṣiṣẹ julọ ni fifọ RNA oni-okun kan.Iṣeduro iṣẹ ṣiṣe iṣeduro jẹ 1-100μG/mL, ibaramu pẹlu ọpọlọpọ awọn eto ifaseyin.Ifojusi iyọ kekere (0-100 mM NaCl) le ṣee lo lati ge RNA ti o ni okun ẹyọkan, RNA ti o ni okun meji, ati awọn ẹwọn RNA ti a ṣẹda nipasẹ isọdọtun RNA-DNA.Bibẹẹkọ, ni ifọkansi iyọ ti o ga (≥0.3 M), RNase A nikan ni pataki RNA ti o ni okun kan.
RNase A jẹ lilo pupọ julọ lati yọ RNA kuro lakoko igbaradi ti DNA plasmid tabi DNA genomic. Boya tabi ko ṣe DNase lọwọ lakoko ilana igbaradi le ni irọrun ni ipa lori iṣesi naa.Ọna ibile ti sise ninu iwẹ omi le ṣee lo lati mu iṣẹ ṣiṣe DNA ṣiṣẹ.Ọja yii ko ni DNAse ati protease ninu, ati pe ko nilo itọju ooru ṣaaju lilo.Ni afikun, ọja yii tun le ṣee lo ni awọn adanwo isedale molikula gẹgẹbi itupalẹ aabo RNase ati itupalẹ ọkọọkan RNA.
Awọn ipo ipamọ
Ọja naa le wa ni ipamọ ni -25 ~-15 ℃, wulo fun ọdun 2.
Awọn ilana
Eyi jẹ ọkan ninu awọn ọna ti o wọpọ fun igbaradi ojutu ibi ipamọ RNase A.O le tun ti wa ni pese sile nipaawọn ọna miiran ni ibamu si awọn ọna ibile ni yàrá tabi awọn iwe itọkasi (biiTutu taara ni 10 mM Tris-HCl, pH 7.5 tabi ojutu Tris-NaCl)
1. Lo 10 mM sodium acetate (pH 5.2) lati mura 10 mg/mL ti RNase ojutu ipamọ
2. Alapapo ni 100 ℃ fun 15 min
3. Dara si iwọn otutu yara, ṣafikun iwọn 1/10 ti 1 M Tris-HCl (pH 7.4), ṣatunṣe pH rẹ si 7.4 (funFun apẹẹrẹ, ṣafikun 500 milimita ti 10g/ml RNase ojutu ipamọ 1M Tris-HCL, PH7.4)
4. Apo-apo ni -20 ℃ fun ibi ipamọ tio tutunini, eyiti o le jẹ iduroṣinṣin fun ọdun 2.
[Awọn akọsilẹ]: Nigbati o ba n ṣatunṣe ojutu RNaseA labẹ awọn ipo didoju, ojoriro RNase yoo dagba;Sise o ni kekere pH, ati pe ti o ba wa ni ojoriro, o le ṣe akiyesi, eyiti o le fa nipasẹ wiwa awọn aiṣedeede amuaradagba.Ti a ba rii erofo lẹhin sise, a le yọ awọn idoti kuro nipasẹ centrifugation iyara giga (13000rpm), ati lẹhinna ṣajọpọ fun ibi ipamọ didi.
ọja alaye
| Awọn itumọ ọrọ sisọ | Ribonuclease I;Pancreatic ribonuclease;Ribonuclease 3'-pyrimidnooligonucleotidohydrolase;Rnase A;Endoribonulcease I |
| CAS No. | 9001-99-4 |
| Ifarahan | Funfun lyophilized lulú |
| Ìwúwo molikula | ~ 13.7kDa (atẹle amino acid) |
| Iye Ph | 7.6 (Iwọn iṣẹ ṣiṣe 6-10) |
| Iwọn otutu to dara | 60℃ (Iwọn iṣẹ ṣiṣe 15-70℃) |
| Aṣoju ṣiṣẹ | Na2+.K+ |
| Inhibitor | Inhibitor Rnase |
| Ọna imuṣiṣẹ | Ko le mu ṣiṣẹ nipasẹ alapapo, o daba lati lo ọwọn centrifuge |
| Ipilẹṣẹ | Bovine |
| Solubility | Tiotuka ninu omi (10mg / milimita) |
| Pipadanu lori gbẹ | ≤5.0% |
| Enzyme aṣayan iṣẹ-ṣiṣe | ≥60 Kunitz sipo/mg |
| Isoelectric ojuami | 9.6 |
Awọn akọsilẹ
Fun ailewu ati ilera rẹ, jọwọ wọ awọn aṣọ laabu ati awọn ibọwọ isọnu fun iṣẹ.
