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TEMED

N,N,N'',N''-四甲基乙二胺

Company: Sigma-Aldrich
Catalog#: T9281
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Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae
Author:
Date:
2020-03-05
[Abstract]  Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O2-) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature of the cofactor that is required for proper activity. Most eukaryotes, including yeast, possess CuZnSOD and MnSOD. This protocol aims at assessing the activity of the yeast Saccharomyces cerevisiae MnSOD Sod2p from cellular extracts using nitroblue tetrazolium staining. This method can be used to estimate the cellular bioavailability of Mn2+ as well as to evaluate the redox state of the cell. [摘要]  [摘要 ] 超氧化物歧化酶(SOD能)充当主防御针对反应性氧物质(ROS)通过转换的超氧阴离子自由基(O 2 - )为分子氧(O 2 )和过氧化氢(H 2 ? 2 )。这种酶的家庭成员包括CuZnSODs ,MnSODs ,FeSODs 和NiSODs ,这取决于是需要适当的活动辅助因子的性质。大多数真核生物,包括酵母,都具有CuZnSOD 和MnSOD 。该协议旨在评估酵母的活性 使用硝基蓝四唑染色法从细胞提取物中提取酿酒酵母MnSOD Sod2p 。该方法可用于估计Mn 2+ 的细胞生物利用度以及评估细胞的氧化还原状态。

[背景 ] 的SODs被定义为减少正常有氧代谢为氧气和过氧化氢期间形成的氧的有害自由基含金属的抗氧化剂酶。:这些酶是基于需要作为辅因子进行适当的酶活性的金属分类CuZnSODs ,MnSODs ,FeSODs ,和NiSODs 。在酿酒酵母中,有两个S OD :CuZn-Sod1p和Mn-Sod2p(Abreu和Cabelli ...

Preparation and Purification of Proteins Secreted from Phytophthora sojae
Author:
Date:
2018-10-20
[Abstract]  Phytophthora sojae, the causal agent of soybean root and stem rot, is responsible for enormous economic losses in soybean production. P. sojae secrets various effectors to reprogram host immunity. The plant apoplastic space is a major battleground in plant-pathogen interactions. Here we describe a protocol for purification and isolation of secreted proteins from P. sojae, including precipitation of secreted proteins from P. sojae culture filtrate, chromatographic purification of the secreted proteins and analysis of the proteins by Mass spectrometry. With this protocol, it will be easier to identify potential apoplastic effectors in Phytophthora and will benefit our understanding of plant-microbe interactions. [摘要]  大豆根和茎腐病的致病因子大豆疫霉(Phytophthora sojae)是造成大豆生产的巨大经济损失的原因。 P上。 sojae >秘密各种效应器重新编程宿主免疫力。植物质外体空间是植物 - 病原体相互作用的主要战场。在这里,我们描述了从 P中纯化和分离分泌蛋白的方案。 sojae >,包括来自 P的分泌蛋白的沉淀。 sojae >培养滤液,分泌蛋白的色谱纯化和质谱分析蛋白质。通过该协议,可以更容易地鉴定 Phytophthora >中潜在的质外体效应物,并有助于我们对植物 - 微生物相互作用的理解。

【背景】从 Phytophthora >物种中分泌的蛋白质的纯化对于理解 Phytophthora >发病机理至关重要。过去,V8果汁和植物(番茄和利马豆)果汁培养基已用于培养 Phytophthora >,培养物过滤后用于分析 Phytophthora >分泌的蛋白质。这些方案的缺点是培养基含有大量的植物蛋白,它们代表了大部分检测到的蛋白质。在该方案中,我们使用了不含任何蛋白质的合成液体培养基。该培养基显着降低了 Phytophthora >培养滤液的背景。此外,利用凝胶过滤脱盐和筛分柱代替离子交换柱,可以有效和大规模纯化 Phytophthora >分泌的蛋白质。

SDS-PAGE for Silk Fibroin Protein
Author:
Date:
2018-10-20
[Abstract]  The method and detailed procedure of SDS-PAGE for silk proteins are exactly the same as for other proteins, but the electrophoresis profile of silk protein is often unsatisfactory. The main reason is that their molecular masses are too large, and the regenerated liquid silk is easily coagulated and denatured, resulting in a significant adverse effect on normal electrophoresis. A satisfactory SDS-PAGE profile for silk protein can be obtained by rapidly loading samples, reducing time and temperature when mixing the sample with the loading dye. [摘要]  丝蛋白的SDS-PAGE方法和详细步骤与其他蛋白质完全相同,但丝蛋白的电泳图谱往往不能令人满意。 主要原因是它们的分子量太大,再生的液体丝很容易凝固和变性,导致对正常电泳的显着不利影响。 通过快速加载样品,在将样品与上样染料混合时减少时间和温度,可以获得令人满意的丝蛋白SDS-PAGE图谱。

【背景】蛋白质或多肽的SDS-PAGE是分析蛋白质亚基分子量的最经典,基本和常用的实验方法之一(Laemmli,1970)。因此,对于大多数蛋白质而言,通常不难获得具有清晰条带的良好SDS-PAGE电泳图谱。

然而,对于参与丝蛋白电泳实验的研究人员来说,似乎不容易获得具有清晰的轻链带(一种丝素蛋白亚基)的良好SDS-PAGE图谱。对于没有长时间电泳经验的初学者或学生来说尤其如此。问题出在哪儿?主要困难与SDS-PAGE技术本身无关,但与丝纤维制备液体丝素蛋白有关,以及丝素蛋白本身的独特性质。

丝蛋白是丝素蛋白和丝胶蛋白的总称。由成熟的家蚕幼虫纺成的两条平行单丝由65%-75%的丝心蛋白,20%-30%的丝胶蛋白和5%的蜡,色素,糖和其他杂质组成。丝素蛋白是一种结晶聚合物基纤维,被几层胶质蛋白包围。丝胶蛋白的最外层易溶于热水或沸水中。最接近丝心蛋白纤维的最内层丝胶蛋白几乎不溶于沸水(Wang和Zhang,2011)。所有分层的丝胶蛋白都易于溶解在碱性热水或沸水中。因此,在实验室中最常使用0.1%-0.5%Na ...

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