| In organello Protein Synthesis
|
|
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 ...
[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。
|
|
|
| Lysosomal Amino Acid Efflux Assay
|
|
Author:
Date:
2014-02-20
[Abstract] As the cellular “recycling” organelle, lysosomes break down proteins into amino acids, which are then transported into cytosol for reuse by various amino acid transporters. The transport rate of an amino acid is presumably regulated by cellular conditions such as organelle pH, membrane potential and metabolic states. Because of their intracellular localization and the relative inaccessibility, lysosomal amino acid transporters have been studied largely via indirect measurements. Using lysosome purification and 14C-labeled amino acids, this protocol provides a method to measure the ...
[摘要] 作为细胞"再循环"细胞器，溶酶体将蛋白质分解成氨基酸，然后将其转运到细胞质中以供各种氨基酸转运蛋白重复使用。 氨基酸的转运速率可能通过细胞条件如细胞器pH，膜电位和代谢状态来调节。 由于它们的细胞内定位和相对不可接近性，溶酶体氨基酸转运蛋白主要通过间接测量进行研究。 使用溶酶体纯化和14 C标记的氨基酸，该协议提供了一种测量特定氨基酸转运蛋白在溶酶体上的效率的方法。
|
|
|