细胞库/细胞培养
ELISA试剂盒
实验室仪器/设备
原辅料包材
体外检测试剂
&&RNase A, DNase and protease-free
RNase A, DNase and protease-free
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DXY All Rights Reserved.RNase A (10mg/ml)/核糖核酸酶A
RNase A (10mg/ml)
&产品编号: ST578
&产品包装: 1ml
&产品价格: 75.00元
产品简介：
&&&&Ribonuclease A，简称RNase A，中文名为核糖核酸酶A。用于消化RNA，进口分装，可直接使用。含微量DNase，在EDTA存在时，并且孵育温度不超过20℃时，检测不到很明显的DNase活性，可以用于质粒和基因组抽提等相关实验中去除RNA。
包装清单：
RNase A (10mg/ml)
保存条件：
&&&&-20℃保存。
注意事项：
&&&&为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用说明：
& 相关产品请点击如下按钮：
使用本产品的相关论文：
1. Wu G, Liu ZS, Qian Q, Jiang CQ.
&&&Medical Journal of Wuhan University.2008 Jan,Vol.29 No.1.
2. Zhao J, Yu H, Lin L, Tu J, Cai L, Chen Y, Zhong F, Lin C, He F, Yang P
J Proteomics. 2011 Dec 21;75(2):588-602.
3. Wang AH, Wei L, Chen L, Zhao SQ, Wu WL, Shen ZX, Li JM.
　 Ann Hematol. ):917-31.
4. Tu Z, Ma Y, Tian J, Li H, Akers W, Achilefu S, Gu Y.
　 J Cancer Res Clin Oncol. ):.
5. Wu J, Zhou Z, Hu Y, Dong S.
　 J Genet Genomics. 2012 Aug 20;39(8):375-84.
6.Nie D, Huang K, Yin S, Li Y, Xie S, Ma L, Wang X, Wu Y, Xiao J.
Leuk Lymphoma. 2012 Jun 18.
7. Zhen-Jun S, Yuan-Yuan Z, Ying-Ying F, Shao-Ju J, Jiao Y, Xiao-Wei Z, Jian C, Yao X,
&& Li-Ming Z.
Biochem Pharmacol. 2012 Aug 15;84(4):507-12.
8. Ling C, Chen G, Chen G, Zhang Z, Cao B, Han K, Yin J, Chu A, Zhao Y, Mao X.
Int J Cancer. 2012 Feb 23.Note: You clicked on an external link, which has been disabled in order to keep your shopping session open.
PureLink& RNase A (20 mg/mL)
PureLink& RNase A (20 mg/mL)
Invitrogen&
&相关应用：
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PureLink& RNase A (ribonuclease A) is a bovine pancreatic ribonuclease that cleaves single-stranded RNA. This reagent is also a component of the PureLink& plasmid purification system.Concentration20 mg RNase A/mL in 50 mM Tris-HCl (pH 8.0), 10 mM EDTA.
For Research Use Only. Not for use in diagnostic procedures.
Product Line:
UltraPure&
Product Size:
Shipping Condition:
Room Temperature
内容及储存
One 10-ml bottleStore at 2&C to 8&C.
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添加至收藏夹RNA analysis
Structural RNAs:
A. Ribosomal RNA analysis
rRNA database project (Max Planck Institute for Marine Microbiology, Bremen, Germany ) - provides comprehensive, quality checked and regularly updated datasets of aligned small (16S/18S, SSU) and large subunit (23S/28S, LSU) ribosomal RNA (rRNA) sequences for all three domains of life (Bacteria, Archaea and Eukarya).
& - predicts 5s/8s, 16s/18s, and 23s/28s ribosomal RNA in full genome sequences (Reference: Lagesen, K et al. 2007. Nucl. Acids Res. 35: )
& (RDP(Michigan State University Centre for Microbial Ecology, U.S.A.)&- provides ribosome related data and services, including online data analysis and aligned and annotated Bacterial and Archaeal small-subunit 16S rRNA sequences.A tutorial is provided .
- Ribosomal RNA analysis for clinically relevant bacteria - (University of W&rzburg, Germany)
- (Universitat Bielefeld, Germany) The RIFLE system compares restriction patterns of 16S rDNA amplicons against a database of theoretical restriction patterns generated from a 16S rDNA database
& - predicts rRNA by using HMMER 3.0 to identify DNA reads containing rRNA sequences. (Reference: S. Wu et al. 2011. BMC Genomics 12:444).
B. Transfer RNAs (tRNA) - for additional information see the genomic tRNA database &or Transfer RNA database
tRNAs:- (Univerisity of California, Santa Cruz, U.S.A,) is incredibly sensitive & also provides secondary structure& diagrams of the tRNA molecules (Reference: Lowe, TM, & Eddy, SR. . 1997. 25: 955-964).&&tRNAscan-SE 2.0&can also be accessed . &Alternatively use
& (Reference: Laslett, D. & Canback. 2004. Nucleic Acids Research 32:11-16) or
- (D. Paul & Md. Aftabuddin, West Bengal University of Technology, India) - identifies tRNA genes without introns or with introns at canonical or non-canonical positions.
& - is a program to detect tRNAs in metazoan mitochondrial DNA sequences (Reference: D. Laslett & B. Canb&ck B. 2008. Bioinformatics 24:172-175)
& - The Rfam database is a collection of RNA families, each represented by multiple sequence alignments, consensus secondary structures and covariance models (Reference: Gardner, P.P. et al. 2008. Nucl. Acids Res. 37, Database issue D136-D140)
& - unlike other tRNA gene-finders, TFAM uses information from the total sequences of tRNAs and not just their anticodons to predict their function. Therefore TFAM has an advantage in predicting initiator tRNAs, the amino acid charging identity of nonstandard tRNAs such as suppressors, and the former identity of pseudo-tRNAs. New TFAM models are available including a proteobacterial model for the bacterial lysylated isoleucine tRNAs, making it now possible for TFAM to correctly classify all tRNA genes for some bacterial taxa.(Reference: H.T&quist et al. 2007. Nucleic Acids Res. 35 (suppl 2): W350-W353).
tmRNAs:&& & - detects transfer-messenger RNAs which function to free ribosome-stalled mRNAs. (Reference: Laslett, D., et al. 2002.& Nucleic Acids Res. 30: , 2002) .
C. Micro RNAs& (miRNAs) are small, non-coding RNA (~20-22 nucleotides) that negatively regulate gene expression at post-transcriptional level.
& -&users can: (i) filter low-quality reads and 3/5' adapters fro (ii) align large-scale short reads to the reference genome and explore their (iii) classify small RNA candidates into known categories, such as known miRNAs, non-coding RNA, genomic repeats
(iv) provide detailed annotation information for known miRNAs, such as miRNA/miRNA*, absolute/relative reads count and t (v) predict novel miRNAs that have not been
and (vi) identify differentially expressed miRNAs between samples based on two different counting strategies.(Reference: Zhu, E.L. et al. 2010. Nucl. Acids Res.&38 (suppl 2): W392-W397).
& -&is a SVM-based software tool for prediction of most probable microRNA coding regions in genome scale sequences (Reference: Wu Y.et al. 2011. BMC Bioinformatics. 12(1):107).
& predict miRNAs from the genomic sequences as well as from Next Generation Sequencing data. It applies a bootstrap aggregating approach to create an ensemble of three different approaches (na&ve Bayes, Best First Decision tree and SVM) to achieve a high accuracy. At present miR-BAG includes 6 different species, 4 for animals (Homo sapiens, Canis familiaris, Mus musculus, Rattus norvegicus) alongwith one nematode (Caenorhabditis elegans) and one insect species (Drosophila melanogaster). miR-BAG was found to perform consistently with accuracy level higher than 90% for several species.(Reference: Jha, A. et al. 2012. PLoS ONE 7(9): e45782.)
&Small nucleolar RNAs (snoRNAs) - can be detected with
for methylation-guide for snoRNAs and
for pseudouridylation-guide snoRNAs (Reference: P. Schattner et al. 2005. Nucl. Acids Res. 33: W686-W689). .
&: an integrated collection of small RNA research tools. Includes: sRNAbench: Expression profiling of small RNAs and prediction of novel microRNAs& from
sRNAde: Differential sRNAblast: Blast analysis of deep sequencing reads against a local nt/nr (NCBI link) database.(Reference: A. Rueda et al. 2015.& Nucl. Acids Res. 43 (W1): W467-W473).
RNA folding:
- Multiple Alignment of RNAs - is a tool for multiple alignment of RNA molecules. LocARNA requires only RNA sequences as input and will simultaneously fold and align the input sequences. LocARNA outputs a multiple alignment together with a consensus structure. For the folding it makes use of a very realistic energy model for RNAs as it is by RNAfold of the Vienna RNA package (or Zuker's mfold). For the alignment it features RIBOSUM-like similarity scoring and realistic gap cost. (Reference: C. Smith et al. 2010. Nucl. Acids Res. 38: W373-377).
is a tool for multiple alignment of RNA molecules. CARNA requires only the RNA sequences as input and will compute base pair probability matrices and align the sequences based on their full ensembles of structures. Alternatively, you can also provide base pair probability matrices (dot plots in .ps format) or fixed structures (as annotation in the FASTA alignment) for your sequences. If you provide fixed structures, only those structures and not the entire ensemble of possible structures is aligned. In contrast to LocARNA, CARNA does not pick the most likely consensus structure, but computes the alignment that fits best to all likely structures simultaneously. Hence, CARNA is particularly useful when aligning RNAs like riboswitches, which have more than one stable structure. (Reference: A. Dragos et al. 2012. Nucleic Acids Reseach 40: W49-W53).&
&&&&&&&&&&&&
- folds and aligns RNA structures (make a foldalignment) based on a lightweight energy model and sequence similarity. The current version makes pairwise fold alignments. (Reference: J. H. Havgaard et al. J. PLOS computational biology. 3:e193, 2007).
For RNA folding use & -& N.B. The data can be presented in a number of graphic formats.& This is my &go to& site if I'm interested in a secondardy structure for a fragment of RNA or DNA (Reference: M. Zuker. 2003. Nucleic Acids Res. 31: ).
& (University of Vienna, Austria). I have found this site useful for drawing tRNAs in cloverleaf format.
is a novel secondary structure prediction method based on conditional log-linear models, a flexible class of probabilistic models which generalize upon stochastic context-free grammars by using discriminative training and feature-rich scoring. By incorporating most of the features found in typical thermodynamic models, CONTRAfold achieves the highest single sequence prediction accuracies to date, outperforming currently available probabilistic and physics-based techniques. It provides MARNA-like output couples with hairpin structures (Reference: Do, C.B. et al. 2006. Bioinformatics 22: e90-e98).
&: a web server and software to design custom and Rfam-based RNA molecules - providing a user-friendly pipeline to design synthetic constructs having the functionality of given Rfam families. In addition, the new software supports amino acid constraints, even for proteins translated in different reading frames from overlapp moreover, structure compatibility/incompatibility constraints have been expanded. With these features, RNAiFold 2.0 allows the user to design single RNA molecules as well as hybridization complexes of two RNA molecules. (Reference: J.A. Garcia-Martin et al. 2015.& Nucl. Acids Res. 43 (W1): W513-W521).
&: a web server for the pairwise global or local alignment of RNA secondary structures. (Reference: E. Mattei et al. 2015.& Nucl. Acids Res.& 43 (W1): W493-W497).
& -& this web server that is capable of superimposing RNA 3D structures by using clique matching and 3D least-squares fitting. Rclick has been benchmarked and compared with other popular servers and methods for RNA structural alignments. In most cases, Rclick alignments were better in terms of structure overlap. It also recognizes conformational changes between structures. (References: Nguyen MN, & Verma C. 2015. Bioinformatics 31:966-968).
Pseudoknots:
RNA/DNA folding predictions including pseudoknots and entangled helices. Provides (i) a series of low free energy structures, (ii) an online animated folding path and (iii) a programmable trajectory plot focusing on a few helices of interest to each user. (Reference: A. Xayaphoummine et al. 2005. Nucl. Acids Res. 33: W605-W6). Structure of GGGAGAUUCCGUUUUCAGUCGGGAAAAACUGAA is shown below:
(Universit&t Bielefeld, Germany) - is a series of 3 tools for folding RNA secondary structures, including the class of simple recursive pseudoknots. Unfortunately to optimally view the results one needs Microsoft.NET framework (massive) and (School of Computer Science and Engineering, Inha University, Korea).
: A Heuristic Approach for Detecting RNA H-type Pseudoknots - offers a variety of tools including pknotsRG, PNOTS and NUPACK (Reference: C.-H. Huang et al. 2005. Bioinformatics 21: ).
& - Predict RNA secondary structures with pseudoknots prediction (Reference: Ren, J. et al. 2005.& RNA 11: ).
&: RNA Pseudoknot Prediction Server (Chiba Institute of Technology, Japan). Offers lots of control on parameters.
& - combines many separate prediction and analysis algorithms: calculating a partition function, predicting a maximum free energy (MFE) structure, finding structures with maximum expected accuracy, and pseudoknot prediction. This server takes a sequence, either RNA or DNA, and creates a highly probable, probability annotated group of secondary structures, starting with the lowest free energy structure and including others with varied probabilities of correctness.
&&- pseudoknot prediction including kissing hairpins - Intramolecular kissing hairpins are a more complex and biologically important type of pseudoknot in which two hairpin loops form base pairs. They are hard to predict using free energy minimization due to high computational requirements. (Reference: Sperschneider J et al.& 2011.& RNA. 17:27-38).
& Provides services for predicting RNA secondary structures including a wide class of pseudoknots. IPknot can also predict the consensus secondary structure when a multiple alignment of RNA sequences is given. IPknot runs fast and predicts the maximum expected accuracy (MEA) structure using integer programming (IP) with threshold cut. (Reference: Satto, K.& et al. 2011. Bioinformatics 27: i85-i93.)
Promoters, terminators and other regulatory elements:
- offers two types of analyses (a) Regulon Analysis - analysis of a whole prokaryotic genome with one regulator pattern and (b) Promoter analysis -& Analysis of a promoter region with several regulator patterns (Reference: R. M&nch et al. 2005. Bioinformatics 2005 21: ).
& - Genome Scanner for Terminators - my favourite terminator search program is finally web enabled.& Please note that if you want to analyze data from a *.gbk file you need to use& their conversion program &GenBank2GeSTer&&first. A complete description of each terminator including a diagram is produced by this program.& This site linked to an extensive database of transcriptional terminators in bacterial genome () (Reference: Mitra A. et al. 2011.& Nucl. Acids Res.
(Softberry Inc.) - is one of&only two tools on the internet for mapping rho-independent terminators. You might consider using the advanced feature options and minimally increase the default energy threshold to -12.0.
& (A. Villegas, Public Health Ontario, Canada) -&an online version of TranstermHP, Reference: Kingsford, C. et al. 2007. Genome Biol. 8: R22) an updated version of TransTerm (Reference: Ermolaeva, M.D. et al.&2000. J Mol Biol301: 27-33)
Riboswitch Explorer - scans &40kb DNA for potential genes (which are linked to BLASTP) and several hundred regulatory elements, including riboswitches. If you click on the &search for attenuators& it finds terminators and antiterminators. (Reference: C. Abreu-Goodger & E. Merino. 2005. Nucl. Acids Res. 33: W690-W692).
& is an integrated web server for identifying functional RNA motifs in an input RNA sequence.& These include Splicing sites ( acceptor site); Splicing regulatory motifs(ESE; ESS; ISE; ISS elements); P Transcriptional motifs (rho-in TRANSFAC); Translational motifs (ribosome binding sites); UTR motifs (UTRsite patterns); mRNA degradation elements (AU-rich elements); RNA editing sites (C-to-U editing sites); Riboswitches (RiboSW); RNA cis-regulatory elements (R ERPIN); Similar functional RNA sequences (fRNAdb); RNA-RNA interaction regions (miRNA; ncRNA). (Reference: Chang TH et al. 2013. BMC bioinformatics 14 Suppl 2:S4).
& - A Regulatory RNA Motifs and Elements Finder - RegRNA is an integrated web server for identifying the homologs of regulatory RNA motifs and elements against an input mRNA sequence. Both sequence homologs and structural homologs of regulatory RNA motifs can be recognized. The regulatory RNA motifs supported in RegRNA are categorized into several classes: (i) motifs in mRNA 5'-untranslated region (5'-UTR) and 3'-UTR; (ii) motifs involved in mRNA (iii) motifs involved in transc (iv) (v) splicing donor/ (vi) and (vii) miRNA target sites.(Reference: Huang HY et al. 2006. Nucleic Acids Res. 34(Web Server issue):W429-34).
- compares existing design tools, including those listed above. They also attempt to improve the MPI principles and existing tools by an algorithm that can filter ineffective siRNAs. The algorithm is based on some new observations on the secondary structure. (Reference: S. M. Yiu et al. (2004) Bioinformatics 21: 144-151).
& (Alignment of RNA Tertiary Structures) - aligns two nucleic acid structures (RNAs or DNAs) in pdb format and detecting apriori unknown common substructures. The identified common substructures can be either large global folds or small local tertiary motifs with at least two successive base pairs. (Reference: O. Dror et al. 2005. Bioinformatics 21 (Suppl 2):ii47-ii53)
& is a tool for sRNA target prediction. It computes whole genome predictions by combination of distinct whole genome IntaRNA predictions (Reference: P.R. Wright et al. 2014. Nucl. Acids Res. 42 (W1), W119-W123).
& - calculates thermodynamic features of sense-antisense hybidization. It predicts the free energy changes& of oligonucleotides binding to a target RNA. It can be used to design efficient siRNA targeting a given mRNA sequence. (Reference: Lu, Z.J. & Mathews, D.H. 2008. Nucleic Acids Res.36:640-647).Rnase vs. Rnase A - 实验交流 - 生物秀
标题: Rnase vs. Rnase A
摘要: Rnase vs
Rnase A请问Rnase和Rnase A使用时有何异同，为何价格差了那么多？还有蛋白酶和蛋白酶k在使用时有何异同? 那个效果更好？第三个问题，为何有的试验提完Genomic DNA后要用一些限制酶切一下，这样做的目的是什么？是否这是提完Genomic DNA后必须要做的？谢谢大家 关键词:[蛋白酶 酶切]……
请问Rnase和Rnase A使用时有何异同，为何价格差了那么多？
还有蛋白酶和蛋白酶k在使用时有何异同? 那个效果更好？
第三个问题，为何有的试验提完Genomic DNA后要用一些限制酶切一下，这样做的目的是什么？是否这是提完Genomic DNA后必须要做的？
谢谢大家回复:
非特异性地降解RNA
RNase A, Bovine Pancreas
核糖核酸酶A(牛胰)：
核糖核酸酶A是内切核糖核酸酶，可特异地攻击RNA上嘧啶残基的3"端，切割与相邻核苷酸形成的磷酸二脂键。反应终产物是嘧啶3"磷酸及末端带嘧啶3"磷酸的寡核苷酸。
来源：牛胰。
DNase活性去除：
将固体RNaseA溶于10mM Tris-HCl，pH7.5，15mM NaCl缓冲液或灭菌水中，在100℃煮沸15min后在室温缓慢冷却。
1. 从DNA：RNA杂交体中除去杂交的RNA区。
2. 确定DNA或RNA中单碱基突变的位置。
单位定义：
在Kunitz测定条件下(25℃，RNA为底物)每分钟使光吸收由A0降到A1所需的酶量为1Kunitz单位，A1为最终光吸收值。
储藏条件：固体RNase A 4℃。回复:
（秦德安）
　　催化蛋白质水解的酶类。种类很多，重要的有胃蛋白酶、胰蛋白酶、组织蛋白酶、木瓜蛋白酶和枯草杆菌蛋白酶等。蛋白酶对所作用的反应底物有严格的选择性，一种蛋白酶仅能作用于蛋白质分子中一定的肽键，如胰蛋白酶催化水解碱性氨基酸所形成的肽键。蛋白酶颁秀广，主要存在于人和动物消化道中，在植物和微中含量丰富。由于动植物资源有限，工业上生产蛋白酶制剂主要利用枯草杆菌、栖土曲霉等微发酵制备。
　　皮革工业的脱毛和软化已大量利用蛋白酶，既节省时间，又改善劳动卫生条件。蛋白酶还可用于蚕丝脱胶、肉类嫩化、酒类澄清。临床上可作药用，如用胃蛋白酶治疗消化不良，用酸性蛋白酶治疗支气管炎，用惮性蛋白酶治疗脉管炎以及用胰蛋白酶、胰凝乳蛋白酶对外科化脓性创口的净化及胸腔间浆膜粘连的治疗。加酶洗衣粉是洗涤剂中的新产品，含碱性蛋白酶，能去除衣物上的血渍和蛋白污物，但使用时注意不要接触皮肤，以免损伤皮肤表面的蛋白质，引起皮疹、湿疹等过敏现象。
Proteinase K
Manual/Protocol Index
Catalog Index
Manual Page | Catalog Page | Tissue Dissociation Guide
--------------------------------------------------------------------------------
I.U.B.: 3.4.21.64 Tritirachium alkaline proteinase
--------------------------------------------------------------------------------
Proteinase K (PROK) is a serine endopeptidase with a broad spectrum of action, isolated from the filtrate of the fungus Tritirachium album limber.
Characteristics of Proteinase K from Tritirachium album limber:
Molecular weight: 28,900 daltons.
pH Optimum: 7.5 - 12, using denatured hemoglobin as substrate.
Stability: Although calcium ions do not affect the enzyme activity, they do contribute to its stability when present at a concentration of 1 - 5 μmoles. An interesting characteristic of proteinase K is that it retains its activity in the presence of sodium dodecyl sulphate (SDS) or urea. (0.5 - 1% SDS and 1 - 4 M urea). Raising the temperature of the reaction from 37°C to 50°C - 60°C can increase the activity several folds. A special feature of proteinase K is its ability to digest native proteins, thereby inactivating enzymes such as DNase and RNase without recourse to a denaturation process.
Specificity: In addition to cleavage of peptide bonds, it is able to catalyze peptide amide hydrolysis. Proteinase K is inactivated by diisopropyl fluorophosphate (DFP) or phenyl methane sulphonyl fluoride (PMSF). Chelating agents such as citrate and EDTA have no affect on the enzyme activity.
Application: Proteinase K is very useful in the isolation of highly native, undamaged DNAs or RNAs, since most microbial or mammalian DNases and RNases are rapidly inactivated by the enzyme, particularly in the presence of 0.5 - 1% SDS.
Storage: Store at 2 - 8°C.
Worthington proteinase K is supplied as a highly purified lyophilized powder. It is tested to be free of DNase and RNase.
Method: Proteinase K hydrolyzes hemoglobin denatured with urea, and liberates Folin Postive amino acids and peptides, which are determined as tyrosine equivalents. 1 unit releases 1 μ mole of Folin positive amino acid in 10 minutes at 37°C, pH 7.5, using denatured hemoglobin as substrate.
0.05 N HCl - Dilute 0.82 ml conc. HCl to 200 ml with reagent grade water.
0.5 M NaOH - Dissolve 4.0 gm NaOH in 200 ml reagent grade water.
Buffer-Substrate - Dissolve 2.0 gm hemoglobin in 35 ml reagent grade water, add 36.0 gm urea and 16 ml 0.5 M NaOH. Stir for 30 - 60 minutes at room temperatue. Add 0.618 gm boric acid and stir. Adjust the pH to 7.5 with 5 N HCl and q.s. to 100 ml.
Tyrosine standard (2.5 nmol/L) - Dissolve 45.3 mg tyrosine in 100 ml of 0.05 N HCl.
0.3 M Trichloroacetic acid - Dissolve 9.8 gm trichloroacetic acid in 200 ml reagent grade water.
Folin Reagent - Add 10 ml Folin-Ciocalteus Phenol Reagent to 20 ml reagent grade water.
Dissolve 10 mg lyophilized material in 1 ml reagent grade water. Prepare a 1:1000 dilution with water immediately before use.
Label clear glass test tubes for blank, standard, and test. Add 2.5 ml buffer-substrate and incubate for 5 minutes at 37°C. Start reaction by adding 0.2 ml tyrosine standard to the standard tube, 0.2 ml of sample to the test, and 0.2 ml of 0.05 N HCl to the blank. Incubate for 10 minutes at 37°C. Stop reaction by the addition of 5.0 ml trichloroacetic acid. Mix, then add 0.2 ml of sample to the blank and standard, and add 0.2 ml of 0.05 N HCl to the test. Mix and let stand for 10 minutes at room temperature, filter and pipette into test tubes 1.0 ml of filtrate, 2.0 ml of 0.5 N NaOH, and 0.6 ml of Folin Reagent. Mix well. Let stand for 15 minutes and read A578 nm.
Calculation
Proteinase K:
Part No. Size
V302B 100mg
Description: Proteinase K, produced by the fungus Tritirachium albumLimber, is a serine protease that exhibits a verybroad cleavage specificity. It cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acidsand is useful for general digestion of protein in biological samples (1). It has been purified to be free of RNase and DNaseactivities. The stability of Proteinase K in urea and SDS and its ability to digest native proteins make it useful for a variety ofapplications, including preparation of chromosomal DNA for pulsed-field gel electrophoresis (2), protein fingerprinting (3,4)
and removal of nucleases from preparations of DNA (5) and RNA (6,7). A typical working concentration for Proteinase K is 50–100&g/ml.
Molecular Weight: 28,900 Daltons.
Reconstitution: Proteinase K is supplied as a lyophilized powder. Reconstitute in 50mM Tris-HCl (pH 8.0), 10mM CaCl2.
Source: Tritirachium album Limber.
Specific Activity: ≥30mAnson units/mg protein. See Product Information Label.
Storage Conditions: The lyophilized powder should be stored desiccated at –20°C, where it is stable for 12 months. The reconstituted protease should be stored at –20°C, where it is stable for 2–3 months. Avoid multiple freeze-thaw cycles or exposure to frequent temperature changes. These fluctuations can greatly alter product stability. It is best to prepare proteasesjust prior to use or aliquot and freeze at –20°C.
I. Reaction Conditions
Protein Denaturation: In general, proteins require denaturation and disulfide bond
cleavage before enzymatic digestion can go to completion. Proteinase K displays strong
proteolytic activity on denatured proteins and on native proteins as well (1).
1. Dissolve 1–10mg of the target protein in 6M guanidine-HCl (or 6–8M urea), 50mM
Tris-HCl (pH 8), 2–5mM DTT (or β-mercaptoethanol) in a reaction volume of up to
1ml (25&l minimum).
2. Heat at 95°C for 15–20 minutes or at 60°C for 45–60 minutes. If smaller amounts of
protein are to be digested, the recommended conditions given can be scaled down
proportionally. However, under no conditions should less than 25&l of the dissolving
agent be used.
3. After denaturation, allow the reaction to cool and add 50mM Tris-HCl (pH 7.5), 5mM
CaCl2 until the guanidine-HCl or urea concentration is below 2M.
Protease Digestion: Add Proteinase K to the reaction to a final concentration of
50–100&g/ml. Incubate at 37–56°C for at least 1 hour. Reducing the temperature to below
37°C will decrease the digestion rate. Longer incubations of up to 24 hours may be
required, depending on the protein. If using longer incubations, one must be very careful
to avoid bacterial contamination.
To terminate the reaction, add an inhibitor of Proteinase K such as PMSF (1) or DFP. The
reaction can also be terminated by the addition of EGTA (pH 8.0) to a final concentration
of 2mM or by TCA precipitation. Proteinase K may not be completely inactivated by EGTA,
as this enzyme retains partial activity in the absence of calcium (7). Heat treatment
(10–15 minutes at 65°C) only partially inactivates Proteinase K (inhibition by no more
than 20–25%).
Protein Cleavage and Nuclease Removal: Proteinase K can be used to cleave
native proteins and to remove nucleases from DNA (5) or RNA (6,7) preparations. If
digestion of a nondenatured (native) protein is desired, incubate the protein with
Proteinase K at a concentration of 50–100&g/ml at 37–56°C in 50mM Tris-HCl (pH 7.5),
5mM CaCl2 or another buffer that is compatible with the stability of the target protein.
To remove nucleases from DNA/RNA preparations, incubate the nucleic acid with
Proteinase K at a concentration of 50&g/ml at 37°C in 0.01M Tris (pH 7.8), 5mM EDTA,
0.5% SDS (7).
Inhibitors: Phenylmethylsulfonyl fluoride (5mM PMSF; 1,8), Diisopropyl phosphorofluoridate
(DFP), EGTA. Proteinase K is not inhibited by EDTA, iodoacetic acid, TLCK or
Stability: Proteinase K is a very stable protease, active in wide pH and temperature
ranges. The protease is active in a pH range of 4.3–12.0, with optimal activity at pH 8.0.
Proteinase K has a broad temperature profile, retaining >80% of its activity at temperatures
of 20–60°C (8). The protease is active in SDS concentrations as high as 0.5%.
Calcium is a stabilizer of Proteinase K; however, when Ca2+ is removed from the enzyme,
20% of the catalytic activity may still remain (7). This may be enough activity to degrade
proteins commonly found in nucleic acid preparations. The enzyme is also active in 1%
(w/v) Triton& X-100 (8).
关于您提的第三个问题，具体的原因我也不太清楚。并不是所有提取DNA后都必须这样做，至少我们试验室不这样做。回复:
非常感谢您的细致回答。
我提Genomic DNA是用来做RAPD和克隆一个功能基因片段的。
从电泳图谱来看，有微量蛋白质存在， RNA倒是有很多。
我想问，微量的蛋白质是否会对后续试验产生严重影响？
由于RNA残留很多，我想处理一下，价格上Rnase和Rnase A差了很多？
便宜的好用吗？两者差别有多大？回复:
1。蛋白质可以通过酚氯仿抽提来去除，然后在经过乙醇沉淀浓缩即可。
2。关于残留的RNA可以使用RNA分解酶处理即可，购买一般的RNASE就可以了。我们一般使用的都是这个产品。
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