蛋白酶抑制剂Cocktail (不含EDTA,100X DMSO储液)

来自 : www.bimake.cn/product/protease 发布时间：2021-03-24

产品优势实验数据价格对比产品描述储存条件实验方法常见问题资料下载 产品优势

成分明晰： 组分清晰、浓度明确、质量可靠，帮助您把不确定性降到最低限度；
保护全面：为所有类型蛋白提供全面的保护，尤其对于大蛋白保护效果显著强于竞品；
使用安全：部分实验室使用PMSF而不是蛋白酶抑制剂Cocktail，但PMSF是剧毒试剂，吸入或皮肤吸收后有致命危险，特别是对孕妇危害更大，而蛋白酶抑制剂Cocktail则使用安全。

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Customer Product Validation(7)

FTO promotes the p-Tau in a TSC-mTOR-dependent manner.(A) Fto knockdown repressed the activation of the mTOR signaling in neurons isolated from 3xTg AD mice. Neurons were infected with indicated lentivirus for 48h.(B) FTO overexpression activated the mTOR signaling in neurons isolated from 3xTg AD mice. Neurons were infected with indicated lentivirus for 48 h.(C) Rapamycin represses FTO-mediated phosphorylation of Tau isolated from 3xTg AD mice. Neurons were infected with indicated lentivirus for 48 h in the presence of rapamycin(100 nM).(D) Fto knockdown increases the total level of TSC1. Neurons from 3xTg AD mice were treated as in (A).(E) FTO overexpression decreases the total level of TSC1. Neurons from 3xTg AD mice were treated as in (B).(F) Fto decreases the mRNA level of Tsc1. Neurons from 3xTg AD mice were treated as in (A) or (B). **p 0.01

(e and f) qRT-PCR and western blot assays showed the altering expression of PTEN after transfection with miRNA mimics (miR-NC, miR-106b mimics and miR-93 mimics) or anti-miRNA (anti-miR-NC, anti-miR-106b and anti-miR-93)

Increase of MMP-13 expression by Microcystin-LR in DLD-1 and HT-29 cells. After 48 h culture with microcystin-LR, the cells were treated with RIPA lysis buffer with 1mMphenylmethylsulfonyl fluoride and protease inhibitor cocktail and the total cellular protein (40 mg/lane) was used for Western blot with a specific anti-MMP-13 antibody. The membrane was probed with an anti-GAPDH antibody to confirm equal loading.

Mol Carcinog, 2015, 10.1002/mc.22298 Cocktail purchased from Bimake

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价格对比 产品描述 背景知识

内源蛋白的产生和降解一般都维持在一个平衡状态，因此在稳定的环境因素下细胞内的蛋白含量是也是稳定的。但是在体外研究中，蛋白合成过程终止，而降解大大增强,从而影响蛋白得率。因此我们建议使用多种小分子组成的抑制剂混合物来抑制蛋白酶的功能，阻止蛋白降解情况的发生。

产品成分 成分 浓度 靶点 AEBSF 104 mM Serine proteases Aprotinin 80 μM Serine proteases Bestatin 5 mM Aminopeptidases E-64 1.5 mM Cysteine proteases Leupeptin 2 mM Serine and cysteine proteases Pepstatin A 1.5 mM Aspartic proteases 产品细节 描述: 6种广谱蛋白酶抑制剂的混合物，保护蛋白完整性 运输条件: 蓝冰运输 注意点: 传统的蛋白酶抑制剂(E-64, AEBSF, bestatin, leupeptin and aprotinin)是不能够抑制某些去泛素化(DUB)蛋白酶(例如ATAXIN-3)的功能的。(参考文献: Neil D. Rawlings, Guy Salvesen et al. Handbook of Proteolytic Enzymes,Vol.1, 2012). 储存条件

产品应保存于-20°C，有效期2年。使用时请室温放置，用完立即放回-20°C。

实验方法 应用领域

蛋白纯化，蛋白免疫印迹, 免疫(共)沉淀，蛋白质pull-down实验，免疫荧光，免疫组织化学，激酶测定等。

使用方法

在室温下溶解DMSO储液， 在您的实验体系中，按照1：100的比率(体积比体积)将抑制剂cocktail加入到样品溶液中(比如细胞裂解液或者组织抽提物)。

常见问题

问题1: 为什么要用蛋白酶抑制剂Cocktail？
回答1: 蛋白酶抑制剂Cocktail是低毒，全面的蛋白保护试剂，最大限度地保护蛋白，使其免于被蛋白酶降解。目前生物学研究日渐深入，更多地将目标锁定为细胞中较为微量的蛋白，如信号通路蛋白、受体蛋白，采用coIP,Pull-down等方法获得样品。对于这些微量的珍贵样品，Cocktail能取得最佳的保护效果，比单一的抑制剂（如PMSF）更为可靠。

问题2: 蛋白酶抑制剂Cocktail能否稀释使用？
回答2: 蛋白酶活性抑制效果和很多因素有关，如目标蛋白降解的难易程度、蛋白酶的浓度、活性、抑制剂的浓度都会影响最后的保护效果。我们的蛋白酶抑制剂Cocktail采用经典浓度配比，确保对不同类型的蛋白酶都有充分的抑制效果。为了取得最好的保护效果，使用时应按照规定比例稀释，这是此类产品的共性。但实际产品里，不同厂商的标示清楚程度不同，有些友商的对稀释操作描述不严谨，很容易产生可以稀释使用的误导，需要仔细辨读，以免影响实验结果。在部分实验室实际应用中，也存在对于已知蛋白的成熟操作流程中减量使用cocktail的情况，对于这类应用，Bimake可对照同类产品的相同比例稀释使用，可以保证保护效果。

问题3: 实验中经常用PMSF来保护蛋白，蛋白酶抑制剂Cocktail和它比有什么优势？
回答3: PMSF是经典的丝氨酸蛋白酶抑制剂，以前被广泛应用于细胞裂解纯化蛋白过程。但由于PMSF在很多方面存在明显的缺点，逐步被保护效果更好，更安全的蛋白酶抑制剂Cocktail所取代。PMSF的毒性较高，因此在蛋白酶抑制剂中选用AEBSF作为其替代品。AEBSF的半致死量是2834mg/kg，而PMSF是200mg/kg，AEBSF安全域值超过PMSF十四倍多，同时有着相当的抑制效果，对于很多丝氨酸蛋白酶的抑制比PMSF更为迅速。PMSF的另一个缺点是易降解失效，它在水溶液中的半衰期是30min，在细胞裂解过程中很容易失效。而蛋白酶抑制剂Cocktail不但保护更加全面，而且效果更加持久。

问题4: 我应该选择哪种类型的蛋白酶抑制剂cocktail， 片剂还是溶液？
回答4: 在选择cocktail产品类型的时候我们需要注意一些细节问题。两种类型的产品都含有添加物。片剂中含有若干种公司专利的未经公开的医药成分， 而溶液型产品中含有溶剂成分DMSO。一般来说这些添加物是不会影响产品效果的。在工业生产中，溶液型产品较片剂来说需要的加工步骤少，因而质量好价格低。而片剂产品在储存和运输两个环节具有优势，片剂能够在4度储存而不需要零下20度。我们推荐使用溶液型产品，价格更低，有效的抑制剂成分含量更高，便于直接分装。

问题5: DMSO会不会对我的实验造成影响？
回答5: DMSO会影响实验结果的可能性很低。在生物科学领域DMSO被作为很多生物活性物质的溶剂，比如蛋白结晶和免疫共沉淀等要求较高的实验体系中DMSO也有应用。DMSO在室温下性质稳定，在低温条件下DMSO还是优秀的细胞冷冻保护剂。

问题6: 用了Cocktail，结果仍然不好是怎么回事？
回答6: 蛋白酶抑制剂Cocktail作用是保护蛋白免受蛋白酶降解威胁，提高蛋白得率。对于绝大多数蛋白，Cocktail都有很好的效果。如果还是出现蛋白得率低的情况，需要谨慎检查实验步骤和方案设计，最大限度发挥Cocktail作用，减少导致蛋白降解的环节。例如，细胞处理前，先要将裂解体系完全准备好，预先加入Cocktail并混匀，将要破碎的细胞，收集完后或从冰箱中取出后立即加入准备好的裂解液中，给予蛋白全程保护。

资料下载

MSDSManual