| Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein
|
|
Author:
Date:
2021-02-20
[Abstract] The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these in vitro experiments help illustrate details about the molecular mechanisms of P-gp, they do not reflect physiological membrane environments in terms of lateral pressure, curvature, constituent lipid species, etc. The protocol presented here includes a detailed guide for analyzing the conformational change of human P-gp in living HEK293 cells by using intramolecular fluorescence resonance energy transfer (FRET), in which excitation of the donor fluorophore is transferred to the acceptor without emission of a photon when two ...
[摘要] [摘要] P-糖蛋白（P-gp；也称为MDR1或ABCB1）的分子机制已主要使用人造膜进行研究，例如脂质去污剂混合胶束，人造脂质双层和源自培养细胞的膜囊泡。尽管这些体外实验有助于阐明有关P-gp分子机制的细节，但它们在侧向压力，曲率，脂质成分等方面并未反映出生理膜环境。 此处提供的协议包括一个详细的指南，该指南用于通过使用分子内荧光共振能量转移（FRET）分析活HEK293细胞中人P-gp的构象变化，其中供体荧光团的激发被转移到受体上而不发射光子当两个荧光蛋白非常接近时。将FRET分析与膜通透性相结合，可以在活细胞中评估小分子（如核苷酸）对构象变化的贡献。
[背景] P-糖蛋白（P-gp）的是ATP驱动药转运该压出各种疏水有毒化合物到细胞外空间。P-gp由形成底物转运途径的两个跨膜结构域（TMD）和结合并水解ATP的两个核苷酸结合结构域（NBD）组成。传输至少需要两个P-gp状态。在向内（药物转运前）构型中，两个NBD分开，两个TMD向细胞内侧开放；在向外（药物转运）构象中，NBD是二聚体的，而TMD在细胞外侧略微开放（Kodan et al。，2020 ）。自从发现P-gp （Juliano和Ling，1976； Chen等，1986； Ueda等，1986 ...
|
|
|
| Total RNA Extraction from Grape Berry Skin for Quantitative Reverse Transcription PCR and Microarray Analysis
|
|
Author:
Date:
2016-04-05
[Abstract] Extraction of high quality RNA is an essential step for quantitative reverse transcription PCR (qRT-PCR) and microarray analysis. However, it is not easy to extract high quality RNA from fruit materials, which contain high amounts of polysaccharides, lipids and secondary metabolites. Wan and Wilkins (1994) had developed ‘Hot Borate Method’ to isolate high quality RNA. Here, we describe a modified protocol of the ‘Hot Borate Method’ to isolate high quality RNA from grape berry skin for qRT-PCR and microarray analysis (Suzuki et al., 2015a; Suzuki et al., 2015b).
[摘要] 高质量RNA的提取是定量逆转录PCR（qRT-PCR）和微阵列分析的必要步骤。 然而，从含有大量多糖，脂质和次级代谢物的水果材料中提取高质量RNA是不容易的。 Wan和Wilkins（1994）开发了"热硼酸盐方法"来分离高质量的RNA。 在这里，我们描述了"热硼酸盐方法"的修饰的协议，从葡萄浆果皮肤中分离高质量的RNA用于qRT-PCR和微阵列分析（Suzuki等人，2015a; Suzuki等人 al。，2015b）。
|
|
|