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首页 >  新闻动态 >  阿拉丁蛋白酶K

阿拉丁蛋白酶K

发布时间:2021-02-05

阿拉丁蛋白酶K

特异性

       蛋白酶K具有广泛的底物特异性。即便存在洗涤剂的情况下,其仍可降解许多非变性状态的蛋白质。蛋白酶K分离自一种可在角质上生长的腐生真菌(Tritirachium album)。因此,蛋白酶K能够降解非变性状态的角质(头发),因而称为“蛋白酶K”[1]。其切割的主要位点为带有封闭氨基基团、邻近脂族或芳香族氨基酸羧基端的肽键。因其广泛的特异性,其较为常用[2,3,4]

生理性质

       蛋白酶K是稳定的S8家族丝氨酸碱性蛋白酶,在邻近活性位点组氨酸的位置含有两个二硫键和一个游离半胱氨酸[2,4]

       分子量:28,930 Da(氨基酸序列)[21];28,500 Da (SDS-PAGE)[22]

       pH范围:7.5-12.0(尿素变性后的血红素为底物),但多数情况下使用的pH范围为7.5-9.0[2,3]

       温度曲线:最适活性温度37℃(20至60℃之间活性>最大活性的80%)[3]

       pI: 8.92

       消光系数:E1%=14.2(280 nm,10mM NaCl和5mM CaCl2,pH8.0[2]

       活化剂:活化需要1-5mM Ca2+。去除酶中的钙离子(加入EDTA)后,会丢失25%的催化活性。但如果通过凝胶过滤去除EDTA-Ca2+复合物,则会总计丢失80%的酶活性,仅在向不含Ca2+的酶中加入过量的Ca2+时,才会发生少量活化[23]

       蛋白酶K在1% TRITON? X-100之中激活,并在0.5% (w/v) SDS之中完全激活。SDS尿素会使蛋白质底物变性,提高消解速率。在这些试剂作用下,蛋白酶K自身的消解速率要慢得多[3,19,20]

       单位定义:在pH7.5、37℃下,每分钟可水解尿素变性的血红素,产生1.0mmole (181mg) 酪氨酸所需的酶量为一单位。

抑制剂

       蛋白酶K的抑制剂为DIFPPMSF(后者使用的终浓度为5 mM)[3] EDTA只会使其部分失活,并不能起到抑制作用。蛋白酶K不会受到碘乙酸、胰蛋白酶特异性抑制剂TLCK糜胰蛋白酶特异性抑制剂TPCK以及对-氯汞基苯甲酸盐抑制。

应用

       *线粒体分离

       *核酸纯化产物的蛋白质消解。在分子生物学应用中,蛋白酶K常用于消解无用的蛋白质,例如从微生物、培养细胞和植物的DNA或RNA制剂中消解核酸酶。[5-11]在核酸制剂之中,这种酶的使用浓度通常为50-200μg/ml,pH7.5-8.0,37℃。孵育时间30分钟至18小时不等。尽管在长期孵育时,蛋白酶K可以自动消解,但通常还是通过后续的本分萃取法使其变性[3]

       *蛋白酶K已经用于去除阳离子蛋白质上结合的内毒素,如溶菌酶和核糖核酸酶A上的内毒素[12]

       *确定酶在膜上的分布[13]

       *处理石蜡包埋的组织,以便暴露抗原结合位点进行抗体标记[14]

       *去除核酸酶进行原位杂交[15]

       *传染性海绵状脑病(TSE)朊病毒研究及草拟的诊断测试方法利用蛋白酶K消解来自大脑组织样本的蛋白质[16,17]

       *采用蛋白酶K消解法进行蛋白酶足迹实验,去除蛋白质-蛋白质表面互作[18]

制备说明

       蛋白酶K可溶于水(1mg/ml),获得无色透明的溶液。

溶解性和溶液稳定性

       建议在-20℃下冻存粉末。产品可稳定保持至少2年。

       蛋白酶K溶液在较广的pH范围内(4.0-12.5,最适pH8.0)保持稳定,同时在使用时可在25-65℃范围内保持稳定。pH8.0时,溶液至少可在4℃下稳定保存12个月[3]。pH4-11.5时,含有Ca2+ (1-6mM)的溶液预计可稳定保存数周。80%的硫酸铵悬液可在4℃下至少稳定保存12个月[2]

产品列表

产品号产品名称规格Cas标准包装
P109033蛋白酶K冻干粉,≥30 units/mg protein39450-01-625mg,100mg,500mg,1g,5g,25g,100g
P128666蛋白酶K 来源于林伯氏白色念球菌≥20 units/mg dry weight39450-01-625mg,100mg,1g
P301575蛋白酶K 来源于林伯氏白色念球菌≥500 units/mL, buffered aqueous glycerol solution39450-01-61ml,5ml,25ml

       阿拉丁是蛋白酶K大规模生产商。我们针对诊断生产和生物技术客户提供了定制配方制剂。详情可咨询官网www.aladdin-e.com

文献列表

[1]Betzel, C., Three Dimensional Structure of Proteinase K at 0.15 nm Resolution. Eur. J. Biochem., 178, 155-171 (1988).

[2]Ebeling, W., et al., Proteinase K from Tritirachium album Linder, Eur. J. Biochem., 47, 91 (1974).

[3]Enzymes of Molecular Biology, vol. 16, Burrell, M.M., ed. Humana Press (Totowa, NJ: 1993), p. 307. Kraus, E., and Femfert, U., Proteinase K from the Mold Tritirachium album Limber, Specificity and Mode of Action. Z. Physiol. Chem., 357, 937 (1976).

[4]Lizardi, P.M., and Engelberg, A., Rapid Isolation of RNA Using Proteinase K and Sodium Perchlorate. Anal. Biochem., 98, 116 (1979).

[5]Gross-Bellard, et al., Isolation of High Molecular Weight DNA from Mammalian Cells, Eur. J. Biochem., 36, 32-38 (1973).

[6]Molecular Cloning: A Laboratory Handbook, 2nd ed., Sambrook et al., eds., Cold Spring Harbor Press (Cold Spring Harbor, NY: 1989) p. 1.61 and p. B.16.

[7]Kasche, V., et al., A Two-step Procedure for Quantitative Isolation of Pure Double-strand DNA from Animal Tissues and Cell Cultures. Prep. Biochem., 11, 233 (1981).

[8]Hansen, J.N., Isolation of Higher Molecular Weight DNA from Bacillus cereus T Using Proteinase K. Prep. Biochem., 4, 473 (1974).

[9]Holm, C., et al., A Rapid, Efficient Method for Isolating DNA from Yeast. Gene, 42, 169 (1986).

[10]La Claire, J.W., and Herrin, D.L., Co-isolation of High-Quality DNA and RNA from Coenocytic Green Algae. Plant Mol. Biol. Reporter, 15, 263 (1997).

[11]Petsch, P., et al., Proteinase K Digestion of Proteins Improves Detection of Bacterial Endotoxins by the Limulus Amebocyte Assay: Application for Endotoxin removal from Cationic Proteins. Anal. Biochem., 259, 42 (1998).

[12]Brdiczyka, D., and Krebs, W., Localization of Enzymes by Means of Proteases. Biochem. Biophys. Acta, 297, 203 (1973).

[13]Short, B.G., et al., Automated Double Labeling of Proliferation and Apoptosis in Glutathione S-transferase-positive Hepatocytes in Rats. J. Histochemistry and Cytochemistry, 45, 1299 (1997).

[14]Angerer, L.M., et al., Identification of Tissue-Specific Gene Expression by in-situ Hybridization. Methods in Enzymology, 152, 649 (1987).

[15]Sakaguchi, S., et al., Accumulation of Proteinase K-Resistant Prion Protein (PrP) is Restricted by the Expression Level of Normal PrP in Mice Inoculated with a Mouse-Adapted strain of the Creutzfeldt-Jakob Disease Agent. J. Virology, 69, 7586 (1995).

[16]Bennion, B.J., and Daggett, V., Protein Conformation and Diagnostic Tests: the Prion Protein. Clinical Chemistry, 48, 2105 (2002).

[17]Hori, R., and Carey, M., Protease Footprinting Analysis of Ternary Complex Formation by Human TFIIA. J. Biol. Chem., 272, 1180 (1997).

[18]Hilz, H., et al., Stimulation of Proteinase K action by Denaturing Agents: Application to the Isolation of Nucleic Acids and the degradation of “Masked” Proteins. Eur. J. Biochem., 56, 103 (1975).

[19]Methods of Enzymatic Analysis, 3rd Edition, Bergmeyer, H.U., ed., Academic Press (New York, NY: 1983) vol. 2, p. 299.

[20]Jany, K.D., et al., Amino Acid Sequence of Proteinase K from the Mold, Tritirachium album Linder. Proteinase K; a Subtilisin-related Enzyme with Disulfide Bonds. FEBS Letters, 199, 139 (1986).

[21]Jany, K.D., and Mayer, B., Proteinase K from Tritirachium album linder, Molecular Mass and Sequence Around the Active Serine Residue. Biol. Chem. Hoppe-Seyler, 366, 485 (1985).

[22]Bajorath, J., et al., The Enzymatic Efficiency of Proteinase K is Controlled by Calcium. Eur. J. Biochem., 176, 441-447 (1988).

[23]IUBMB Enzyme Nomenclature: http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/64.html


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