Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates.
Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates.
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DOI：
10.1186/1472-6750-11-125
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发表时间：
2011-12-19
影响因子：
3.5
通讯作者：
Webb WW
中科院分区：
文献类型：
--
作者：
Anderson VL;Webb WW
荧光标签，包括有机小分子和荧光蛋白，可以在生物医学研究实验中定位蛋白质分子。然而，这些标记的使用可能会干扰更大规模的蛋白质结构（例如淀粉样蛋白聚集体）的形成。因此，我们研究了一些常用荧光标签对阿尔茨海默病相关肽淀粉样蛋白 β 1-40 (Aβ40) 和帕金森病相关蛋白 α-突触核蛋白 (αS) 生长的原纤维形态的影响。使用透射电子显微镜 (TEM)，我们验证了 AMCA、TAMRA 和 Hilyte-Fluor 488 标签对 Aβ40 的 N 端标记不会阻止各种宽度的原纤维和淀粉样原纤维的形成。我们还测量了 Hilyte Fluor 488 标记的 Aβ40 的双光子作用横截面，并证明该标签适合与双光子荧光技术一起使用。同样，我们发现 Alexa Fluor 488 对 N 或 C 末端（位置 9 或 130）附近的 αS 变异蛋白进行标记不会干扰淀粉样蛋白和其他类型 αS 原纤维的形成。我们还展示了从用增强型绿色荧光蛋白 (EGFP) 标记的 αS C 末端生长的原纤维的 TEM 图像。在接近中性 pH 值时，通过 TEM 观察到两种类型的 αS-EGFP 原纤维，而 EGFP 标签的变性导致其他物种的形成。我们证明了几种小的外在荧光标签与淀粉样蛋白聚集的研究相容。然而，虽然原纤维可以从用 EGFP 标记的 αS 中生长出来，但荧光蛋白标签的构象会影响观察到的聚集体形态。因此，我们的结果应该有助于研究人员进行涉及荧光技术的聚集研究的标签选择和解决方案条件优化。
Fluorescent tags, including small organic molecules and fluorescent proteins, enable the localization of protein molecules in biomedical research experiments. However, the use of these labels may interfere with the formation of larger-scale protein structures such as amyloid aggregates. Therefore, we investigate the effects of some commonly used fluorescent tags on the morphologies of fibrils grown from the Alzheimer's disease-associated peptide Amyloid β 1-40 (Aβ40) and the Parkinson's disease-associated protein α-synuclein (αS). Using transmission electron microscopy (TEM), we verify that N-terminal labeling of Aβ40 with AMCA, TAMRA, and Hilyte-Fluor 488 tags does not prevent the formation of protofibrils and amyloid fibrils of various widths. We also measure the two-photon action cross-section of Aβ40 labelled with Hilyte Fluor 488 and demonstrate that this tag is suitable for use with two-photon fluorescence techniques. Similarly, we find that Alexa Fluor 488 labelling of αS variant proteins near either the N or C terminus (position 9 or 130) does not interfere with the formation of amyloid and other types of αS fibrils. We also present TEM images of fibrils grown from αS C-terminally labelled with enhanced green fluorescent protein (EGFP). Near neutral pH, two types of αS-EGFP fibrils are observed via TEM, while denaturation of the EGFP tag leads to the formation of additional species. We demonstrate that several small extrinsic fluorescent tags are compatible with studies of amyloid protein aggregation. However, although fibrils can be grown from αS labelled with EGFP, the conformation of the fluorescent protein tag affects the observed aggregate morphologies. Thus, our results should assist researchers with label selection and optimization of solution conditions for aggregation studies involving fluorescence techniques.
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影响因子：
15
作者：
Schierle, Gabriele S. Kaminski;van de Linde, Sebastian;Kaminski, Clemens F.
通讯作者：
Kaminski, Clemens F.
DOI：
10.1016/j.bbadis.2008.06.003
发表时间：
2008-09-01
影响因子：
6.2
作者：
Chafekar, Sidhartha M.;Baas, Frank;Scheper, Wiep
通讯作者：
Scheper, Wiep
影响因子：
4.4
作者：
Neu, TR;Kuhlicke, U;Lawrence, JR
通讯作者：
Lawrence, JR
DOI：
10.1073/pnas.96.7.3590
发表时间：
1999-03-30
影响因子：
11.1
作者：
Chiti, F;Webster, P;Dobson, CM
通讯作者：
Dobson, CM
影响因子：
5.6
作者：
van Ham, Tjakko J.;Esposito, Alessandro;Bertoncini, Carlos W.
通讯作者：
Bertoncini, Carlos W.