| Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System
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Author:
Date:
2020-03-05
[Abstract] T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand interaction durations impacted on T cell activation. Our newly created opto-ligand-TCR system allowed us to precisely and reversibly control ligand binding to the TCR by light illumination. This system uses phytochrome B (PhyB) tetramers as a light-regulated TCR ligand. PhyB can be photoconverted between a binding (ON) and non-binding (OFF) conformation by 660 nm and 740 nm light illumination, respectively. PhyB ON is able to bind to a synthetic ...
[摘要] [摘要] T细胞是免疫系统的一种主要细胞类型，利用其T细胞抗原受体（TCR）结合并响应源自病原体的外来分子。配体-TCR相互作用的半衰期决定了刺激的结果。直到最近，科学家还是依靠突变TCR或其配体来研究变化的TCR-配体相互作用持续时间如何影响T细胞活化。我们新创建的光配体-TCR系统使我们能够通过光照精确且可逆地控制配体与TCR的结合。该系统使用植物色素B（PhyB ）四聚体作为光调节的TCR配体。PHYB 可光转化的结合（ON）之间，并且通过分别为660nm和740nm的光照射，非结合性（OFF）构象。PhyB ON能够结合通过将PhyB 相互作用因子（PIF）融合到TCRβ链而产生的合成TCR 。将PhyB 切换为OFF构象会破坏这种相互作用。PhyB 四聚体与PIF-TCR 足够长的结合导致T细胞活化（通过钙内流测量）。在这里，我们描述了有关如何为我们的光配体-TCR系统生成四聚体配体，如何通过流式细胞仪测量配体-TCR结合以及如何通过钙内流量化T细胞活化的协议。
[背景 ] ...
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| In organello Protein Synthesis
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Author:
Date:
2014-06-20
[Abstract] In organello protein synthesis method allows the analysis of mitochondrial translation products. The principle of this method relies on incubation of isolated intact mitochondria with radiolabeled amino acids such as 35S methionine. After protein synthesis, the radiolabeled translation products are subsequently separated by SDS polyacrylamide gel electrophoresis and analysed by autoradiography. For in organello analysis of protein synthesis, the isolated intact mitochondria must retain their bioenergetics capacity, and in consequence be fully functional and able to perform coupled respiration. This in turn requires a quick and gentle purification of mitochondria during their isolation.
[摘要] 在organello中，蛋白质合成方法允许分析线粒体翻译产物。 该方法的原理依赖于分离的完整线粒体与放射性标记的氨基酸如35 S-甲硫氨酸的孵育。 在蛋白质合成之后，随后通过SDS聚丙烯酰胺凝胶电泳分离放射性标记的翻译产物，并通过放射自显影进行分析。 对于蛋白质合成的分析，分离的完整线粒体必须保持其生物能量能力，因此是完全功能的并且能够进行耦合呼吸。 这反过来需要在其分离期间快速和温和地纯化线粒体。
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