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D-(+)-Glucose

D-(+)-葡萄糖

Company: Sigma-Aldrich
Catalog#: G8270
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Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803
Author:
Date:
2018-06-20
[Abstract]  Metals are essential in many biological processes, including oxygenic photosynthesis. Here we described a method to measure the metal pool in whole cells and thylakoids, including the bioactive pool in intact photosynthetic protein complexes in the model oxygenic cyanobacterium Synechocystis PCC6803. In the first part of the protocol, whole cells and thylakoid membranes are carefully prepared, in which the total metal concentrations are measured by inductively coupled plasma triple-quadrupole mass spectrometry (ICP-QQQ-MS). In the second part of the protocol, isolated thylakoids are solubilized to release the integral membrane proteins and the metal binding protein complexes. These intact photosynthetic protein complexes are subjected to size exclusion chromatography (SEC) and ... [摘要]  金属在许多生物过程中是必不可少的,包括含氧光合作用。 在这里我们描述了测量全细胞和类囊体中金属库的方法,包括模型含氧蓝藻PCH6803中完整光合蛋白复合体中的生物活性库。 在方案的第一部分中,仔细制备全细胞和类囊体膜,其中通过电感耦合等离子体三重四极杆质谱(ICP-MS / MS)测量总金属浓度。 在该方案的第二部分,分离的类囊体被溶解以释放完整的膜蛋白和金属结合蛋白复合物。 将这些完整的光合蛋白复合物进行尺寸排阻色谱(SEC),并通过ICP-MS / MS进行连接分析尺寸分离的复合物中的金属结合。

【背景】氧光合作用的过程需要金属,因为它们在光合电子传递链中作为辅因子和催化剂的基本功能。光合装置需要Fe-S簇,血红素桥Fe和非血红素Fe形式的铁(Fe),可溶性移动电子载体蛋白质质体蓝素中的铜(Cu),叶绿素中的镁(Mg), ...

Dual-probe RNA FRET-FISH in Yeast
Author:
Date:
2018-06-05
[Abstract]  mRNA Fluorescence In Situ Hybridization (FISH) is a technique commonly used to profile the distribution of transcripts in cells. When combined with the common single molecule technique Fluorescence Resonance Energy Transfer (FRET), FISH can also be used to profile the co-expression of nearby sequences in the transcript to measure processes such as alternate initiation or splicing variation of the transcript. Unlike in a conventional FISH method using multiple probes to target a single transcript, FRET is limited to the use of two probes labeled with matched dyes and requires the use of sensitized emission. Any widefield microscope capable of sensitive single molecule detection of Cy3 and Cy5 should be able to measure FRET in yeast cells. Alternatively, a FRET-FISH method can be ... [摘要]  mRNA荧光原位杂交(FISH)是一种常用于分析细胞中转录物分布的技术。 当与常见的单分子技术荧光共振能量转移(FRET)相结合时,FISH也可用于分析转录本中附近序列的共表达以测量转录本的替代启动或剪接变异等过程。 与使用多个探针靶向单个转录物的常规FISH方法不同,FRET限于使用用匹配染料标记的两个探针,并且需要使用敏化发射。 任何能够灵敏地检测Cy3和Cy5单分子的宽视场显微镜应该能够测量酵母细胞中的FRET。 或者,可以使用FRET-FISH方法明确确定转录本的身份,而不使用其他FISH技术中使用的引导探针组。

【背景】单细胞转录物分布的定量通常通过用多个探针靶向mRNA来实现,以实现可以与非特异性结合的探针区分开的明亮信号(Raj和Tyagi,2010)。但是,在某些情况下,转录本上有特征,例如剪接变体或替代起始位点,这与常规FISH探针组无法区分。这些同种型序列可以具有短的50nt唯一识别序列。使用两种探针,可以使用FRET对定位结合的任一侧,同时定量多达三种mRNA同种型,例如,具有两种探针的同种型(FRET),具有探针1的同种型仅限于探针2的同种型。依赖于单个荧光团或荧光团对需要通过EMCCD进行灵敏检测。而且,可以使用FRET对(Wadsworth等人,2017)来估计没有其他同种型的序列的探针的检测效率。

Single-probe RNA FISH in Yeast
Author:
Date:
2018-06-05
[Abstract]  Quantitative profiling of mRNA expression is an important part of understanding the state of a cell. The technique of RNA Fluorescence In Situ Hybridization (FISH) involves targeting an RNA transcript with a set of 40 complementary fluorescently labeled DNA oligonucleotide probes. However, there are many circumstances such as transcripts shorter than 200 nt, splicing variations, or alternate initiation sites that create transcripts that would be indistinguishable to a set of multiple probes. To this end we adapted the standard FISH protocol to allow the use of a single probe with a single fluorophore to quantify the amount of transcripts inside budding yeast cells. In addition to allowing the quantification of short transcripts or short features of transcripts, this technique ... [摘要]  mRNA表达的定量分析是理解细胞状态的重要部分。 RNA荧光原位杂交(FISH)技术涉及用一组40个互补的荧光标记的DNA寡核苷酸探针靶向RNA转录物。 然而,许多情况下,如转录本短于200 nt,剪接变异,或创建转录本的替代起始位点,这些转录本与一组多重探针无法区分。 为此,我们调整了标准FISH方案,以允许使用具有单个荧光团的单个探针来量化出芽酵母细胞内的转录物的量。 除了允许定量短转录本或转录本的短特征之外,该技术还降低了执行FISH的成本。

【背景】通过单分子荧光原位杂交(smFISH)可以精确定量单细胞转录谱。 该过程通过用多个荧光标记的DNA寡核苷酸探针靶向单个mRNA分子给出了良好的噪声信号(Raj和Tyagi,2010)。 使用该方案,不能检测到长度短于200个核苷酸的mRNA。 然而,在大多数实验中,绝对转录本拷贝数比相对拷贝数少。 为了检测短的转录物或序列,可以使用短的单个DNA寡核苷酸探针。 当使用单个荧光团计数mRNA时,单个探针的检测效率大于50%(Wadsworth等人,2017)。

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