通过扩散分析剂扩散和抗体结合放大来增强单细胞西部血栓敏感性
Mariia Alibekova Long1,2,3, William K J Benman1,2, Nathan Petrikas1
1Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
Analytical chemistry
|November 17, 2023
概括
我们增强了单细胞西斑 (scWestern) 敏感性,以检测低丰度蛋白质. 通过将斑点转移到酸纤维素并使用酶-抗体结合物,我们实现了检测极限的120倍改进.
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:
- 单细胞分析对于理解细胞异质性至关重要.
- 目前用于单细胞的蛋白质检测方法往往缺乏灵敏度和吞吐量.
- 单细胞西部斑块 (scWesterns) 具有潜力,但由于对低丰度蛋白质的敏感性而受到限制.
研究的目的:
- 为了提高单细胞西部斑点 (scWesterns) 的灵敏度和检测极限.
- 为了能够在单细胞中检测出低丰度蛋白质.
- 通过启用新的检测策略来扩大scWesterns的实用性.
主要方法:
- 通过将scWesterns转移到酸纤维素,从检测介质中进行脱电泳分离.
- 利用酶-抗体结合物来增强对被抹掉的蛋白质的探测.
- 将检测效率与传统的内凝探测方法进行比较.
主要成果:
- 通过将scWesterns转移到酸纤维素来实现检测极限的5.9倍改善.
- 通过使用酶-抗体结合物,进一步提高了检测极限到1000个分子 (120倍的增加).
- 与凝内检测相比,EGFP表达细胞的检测率从84.5%增加到100%.
结论:
- 用酸纤维素固定的scWestern显著提高了蛋白质检测灵敏度.
- 改进的方法允许检测以前无法获得的低丰度蛋白质.
- 这种技术扩大了用于单细胞蛋白质分析的兼容亲和反应物的范围.
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