一个快速和的TMP-AcBOPDIPY探针用于对活细胞中的蛋白质进行共价标记
1Chemical Genomics Centre of the Max Planck Society , Otto-Hahn-Str. 15, 44227 Dortmund, Germany.
Journal of the American Chemical Society
|March 7, 2014
概括
我们开发了一种新的TMP-AcBOPDIPY探针,用于在活细胞中快速和选择性标记蛋白质. 这种化学标记方法可以使用光显微镜可视化细胞内过程.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 细胞生物学 细胞生物学
背景情况:
- 蛋白质标签对于理解细胞和生物的蛋白质功能至关重要.
- 化学标记为活细胞应用提供了新一代蛋白质标记策略.
研究的目的:
- 开发一种新的多功能探针,用于在活细胞中选择性和开启性蛋白质标记.
- 为了创建一个快速和高效的化学探针,用于蛋白质的表征.
主要方法:
- 开发一种TMP-AcBOPDIPY探针,通过将Trimethoprim (TMP) 与一个具有烯胺组的BOPDIPY光体结合起来.
- 一个小的单体标签,大肠杆菌二叶酸减少酶 (eDHFR) 的工程,具有战略位置的氨酸残留物.
- 使用亲和联来快速标记工程化EDHFR标签.
主要成果:
- 该TMP-AcBOPDIPY探测器证明了eDHFR标签的快速标记,其半衰期约为2分钟.
- 探测器在标记蛋白质时显示出2倍的光增强.
- 在活细胞中实现了细胞内蛋白质的特定标记,无论是用染料洗还是不用.
- 通过光终身成像显微镜 (FLIM) 成功可视化细胞内过程.
结论:
- 开发的TMP-AcBOPDIPY探针是一种高效和选择性的活细胞蛋白质标记工具.
- 这种方法有助于研究活细胞内的蛋白质功能和动态.
- 探测器在FLIM中的性能证明了其在先进的蜂成像应用中的潜力.
相关概念视频
03:31Labeling DNA Probes
7.7K
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
7.7K
01:19Protein Dynamics in Living Cells
1.9K
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
1.9K