| Activity-based Pull-down of Proteolytic Standard and Immunoproteasome Subunits
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Author:
Date:
2016-12-20
[Abstract] Activity-based probes (ABP) are small organic molecules that irreversibly bind to the active center of a specific enzyme family and may be coupled to a fluorophore or an affinity tag (Li et al., 2013). Here, we describe a method to pull-down active catalytic standard and immunoproteasome subunits in cell lysates using the biotinylated, proteasome-specific ABP Biotin-Epoxomicin (Bio-EP). Covalent labeling of the active catalytic subunits with Bio-EP is followed by a pull-down using streptavidin-coated beads. After elution from the beads, enriched subunits may be detected via Western blot, tandem mass spectrometry (Li et al., 2013), or alternative techniques.
[摘要] 基于活性的探针（ABP）是不可逆地结合特定酶家族的活性中心并且可以偶联至荧光团或亲和标签的小有机分子（Li等人，2013）。在这里，我们描述了使用生物素化的蛋白酶体特异性ABP生物素 - 环氧丙素（Bio-EP）在细胞裂解物中下拉活性催化标准和免疫蛋白酶体亚基的方法。用Bio-EP共价标记活性催化亚单位，然后使用链霉抗生物素蛋白包被的珠子进行下拉。从珠中洗脱后，可以通过Western印迹，串联质谱法（Li et al。，2013）或其他技术检测富集的亚单位。
背景 蛋白酶体是存在于真核细胞的细胞核和细胞质中的桶形多分子酶复合物。蛋白质降解是重要的，包括加工MHC I呈递的抗原肽，并调节许多细胞过程（Kammerl和Meiners，2016）。在造血起源的细胞中，标准（组成型）蛋白酶体通常被免疫蛋白酶体（Meiners等人，2014）所替代，其在三种不同的催化活性β-亚基的掺入中不同（图1）。
为了研究单个催化亚基的分子功能并调节生理过程，亚基特异性蛋白酶体抑制剂的发展是必不可少的。 de ...
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| Detection of Protein Oxidative Activity Using Reduced RNase A
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Author:
Date:
2016-03-20
[Abstract] This assay allows to determine whether proteins possess oxidative activity-the ability to introduce disulfide bond in vitro. The substrate for potential oxidases is a ribonuclease A which, for its activity, needs 4 properly formed disulfide bonds (Raines, 1998).
RNase A activity can be detected by:
- Monitoring the digestion of RNA (Lambert and Freedman, 1983);
- Methylene Blue assay (Greiner-Stoeffele et al., 1996);
- Analyzing the cleavage of the cyclic CMP (Lyles and Gilbert, 1991; Lyles and Gilbert, 1991).
We here describe method for measurements of oxidative activity, based on the cleavage of cCMP.
Oxidative activity will be tested by measuring spectrophotometrically RNase A cleavage of cyclic-2’, ...
[摘要] 该测定允许确定蛋白质是否具有氧化活性 - 在体外引入二硫键的能力。 潜在氧化酶的底物是核糖核酸酶A，其活性需要4个适当形成的二硫键（Raines，1998）。
核糖核酸酶A活性可通过以下检测：
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| Extraction of Chloroplast Proteins from Transiently Transformed Nicotiana benthamiana Leaves
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Author:
Date:
2014-09-20
[Abstract] This rapid protocol allows the extraction of chloroplast enriched proteins from Nicotiana benthamiana (N. benthamiana) leaves that were transiently transformed to express an epitope tagged protein of interest. Thus, it can serve as a tool to study the chloroplastidic localization of the protein of interest when it is combined with western-blot analysis.
Agrobacterium-mediated transformation (Agroinfiltration, Romeis et al., 2001) is used to transiently express a protein carrying an epitope tag in tobacco leaves. Here, co-infiltration with an Agrobacterium strain harboring 19 K from soil-borne wheat mosaic virus suppresses posttranscriptional gene silencing and therefore increases transformation efficiency (Te et al., 2005). ...
[摘要] 该快速方案允许从瞬时转化以表达感兴趣的表位标记蛋白的本氏烟草（本氏烟草）叶中提取富含叶绿体的蛋白。因此，当它与Western印迹分析结合时，它可以作为研究目标蛋白质的叶绿体定位的工具。使用土壤杆菌介导的转化（Agroinfiltration，Romeis等人，2001）瞬时表达在烟草叶中携带表位标签的蛋白质。在这里，与土壤传播的小麦花叶病毒携带19K的农杆菌菌株的共浸润抑制转录后基因沉默，并因此增加转化效率（Te等人，2005） 。转化的叶子的叶绿体分离基于Romeis等人（2001）的修改，包括细胞壁和膜的机械破坏，通过过滤去除未破坏的组织，通过Percoll层离心分离完整叶绿体。
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