Multiple Sampling in Single-Cell Enzyme Assays Using CE-Laser-Induced Fluorescence to Monitor Reaction Progress

化学 荧光 采样(信号处理) 激光诱导荧光 分析化学(期刊) 生物物理学 色谱法 生物化学 光学 物理 生物 探测器
作者
Glen K. Shoemaker,Justin L. Lorieau,Leon H. Lau,C. Stewart Gillmor,Monica M. Palcic
出处
期刊:Analytical Chemistry [American Chemical Society]
卷期号:77 (10): 3132-3137 被引量:33
标识
DOI:10.1021/ac0481304
摘要

A novel method for assaying enzymes from a single cell or small cell populations is described. The key advantage of this method is the ability to repeatedly sample a single cell enzyme reaction. Whereas multiple sampling has been achieved for larger cell types with a diameter of 1 mm, we report a technique by which single cell enzyme assays of small cells (15 μm in diameter) can be repeatedly carried out. Individual cells were isolated using an in-house-built micromanipulator and placed in nanoliter-scale reaction vessels. The cells were lysed with solution containing substrate, and enzyme activity was assayed by removing 5-nL aliquots with a recently developed nanopipettor. The reaction aliquot was then analyzed using capillary electrophoresis with laser-induced fluorescence detection to quantitate enzyme activity. Sf9 cells were assayed at the single cell level and found to be highly heterogeneous with respect to α-glucosidase II activity. Since only 5 nL of the single cell reaction was removed, multiple sampling was possible, allowing triplicate analysis of enzyme activity for each individual cell. Multiple sampling also permitted a single cell reaction to be monitored over time. The sensitivity of this method was demonstrated in the analysis of a low-abundance enzyme, α1,3-N-acetylgalactosaminyltransferase, from single HT29 cells. Detecting the product of this enzyme reaction required minimizing the dilution of cellular contents. To demonstrate the potential applications of this methodology in small scale biochemical analyses, single Arabidopsis knf embryos lacking the α-glucosidase I encoding KNOPF gene were assayed. Mutant embryos demonstrated insignificant conversion of a triglucose substrate, as compared to wild type, confirming the deletion of α-glucosidase I. Embryos were simultaneously assayed for a second enzyme, β-galactosidase, illustrating that the mutants were viable except for their lack of α-glucosidase I activity.
最长约 10秒,即可获得该文献文件

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
大幅提高文件上传限制,最高150M (2024-4-1)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
叶痕TNT完成签到 ,获得积分10
9秒前
薏仁完成签到 ,获得积分10
11秒前
xiying完成签到 ,获得积分10
12秒前
王胖发布了新的文献求助10
16秒前
19秒前
温暖完成签到 ,获得积分10
24秒前
现实的曼安完成签到 ,获得积分10
25秒前
liner完成签到 ,获得积分10
33秒前
Ning00000完成签到 ,获得积分10
36秒前
鞑靼完成签到 ,获得积分10
39秒前
蓝眸完成签到 ,获得积分10
39秒前
迷路迎南完成签到 ,获得积分10
43秒前
haochi完成签到,获得积分10
44秒前
西西完成签到 ,获得积分10
55秒前
onevip完成签到,获得积分10
1分钟前
swordshine完成签到,获得积分10
1分钟前
哈哈完成签到 ,获得积分10
1分钟前
友好的牛排完成签到,获得积分10
1分钟前
猴子请来的救兵完成签到,获得积分10
1分钟前
Cold-Drink-Shop完成签到,获得积分10
1分钟前
niuya完成签到,获得积分10
1分钟前
ni完成签到 ,获得积分10
1分钟前
轩辕剑身完成签到,获得积分10
1分钟前
蚂蚁踢大象完成签到 ,获得积分10
1分钟前
飞翔的企鹅完成签到,获得积分10
1分钟前
高速旋转老沁完成签到 ,获得积分10
1分钟前
康复小白完成签到 ,获得积分10
1分钟前
小王完成签到 ,获得积分10
1分钟前
zijinbeier完成签到 ,获得积分10
1分钟前
西西弗完成签到 ,获得积分10
1分钟前
明天更好完成签到 ,获得积分10
1分钟前
诺亚方舟哇哈哈完成签到 ,获得积分0
1分钟前
猪仔5号完成签到 ,获得积分10
2分钟前
居里姐姐完成签到 ,获得积分10
2分钟前
牛奶面包完成签到 ,获得积分10
2分钟前
2分钟前
王大橘完成签到 ,获得积分10
2分钟前
舒适的涑完成签到 ,获得积分10
2分钟前
xiazhq完成签到,获得积分10
2分钟前
三伏天完成签到,获得积分10
2分钟前
高分求助中
Spray / Wall-interaction Modelling by Dimensionless Data Analysis 2000
ALA生合成不全マウスでの糖代謝異常の分子機構解析 520
安全防范技术与工程 500
Mathematics and Finite Element Discretizations of Incompressible Navier—Stokes Flows 500
2024 Medicinal Chemistry Reviews 400
Актуализированная стратиграфическая схема триасовых отложений Прикаспийского региона. Объяснительная записка 360
Dictionary of socialism 350
热门求助领域 (近24小时)
化学 医学 生物 材料科学 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 基因 遗传学 催化作用 物理化学 免疫学 量子力学 细胞生物学
热门帖子
关注 科研通微信公众号,转发送积分 3192705
求助须知:如何正确求助?哪些是违规求助? 2841742
关于积分的说明 8034584
捐赠科研通 2505438
什么是DOI,文献DOI怎么找? 1338603
科研通“疑难数据库(出版商)”最低求助积分说明 638372
邀请新用户注册赠送积分活动 606929