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
赵银志完成签到 ,获得积分10
刚刚
无极2023完成签到 ,获得积分10
1秒前
巫巫巫巫巫完成签到 ,获得积分10
2秒前
123456777完成签到 ,获得积分10
3秒前
桃儿完成签到 ,获得积分10
13秒前
李思晴完成签到 ,获得积分10
14秒前
yeape完成签到,获得积分10
15秒前
轩辕剑身完成签到,获得积分10
20秒前
咯咯咯完成签到 ,获得积分10
22秒前
25秒前
zxvcbnm发布了新的文献求助10
29秒前
安安完成签到 ,获得积分10
30秒前
张颖完成签到 ,获得积分10
35秒前
zxvcbnm完成签到,获得积分10
36秒前
onevip完成签到,获得积分10
38秒前
美丽依波完成签到 ,获得积分10
40秒前
三脸茫然完成签到 ,获得积分10
46秒前
17852573662完成签到,获得积分10
46秒前
人类繁殖学完成签到 ,获得积分10
49秒前
别具一格完成签到 ,获得积分10
49秒前
dent强完成签到 ,获得积分10
56秒前
XMUZH完成签到 ,获得积分10
1分钟前
天真的莺完成签到,获得积分10
1分钟前
哆小咪完成签到 ,获得积分10
1分钟前
wBw完成签到,获得积分10
1分钟前
1分钟前
橄榄囚徒完成签到 ,获得积分10
1分钟前
雪山飞龙发布了新的文献求助30
1分钟前
科研小白完成签到 ,获得积分10
1分钟前
文献搬运工完成签到 ,获得积分10
1分钟前
只喝白开水完成签到 ,获得积分10
1分钟前
杨永佳666完成签到 ,获得积分10
1分钟前
1分钟前
fireking_sid完成签到,获得积分10
1分钟前
伊yan完成签到 ,获得积分10
1分钟前
2分钟前
王王完成签到 ,获得积分10
2分钟前
奋斗的妙海完成签到 ,获得积分0
2分钟前
蚂蚁踢大象完成签到 ,获得积分10
2分钟前
chrysan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
Aspects of Babylonian celestial divination: the lunar eclipse tablets of Enūma Anu Enlil 500
Mathematics and Finite Element Discretizations of Incompressible Navier—Stokes Flows 500
Dictionary of socialism 350
Mixed-anion Compounds 300
Geochemistry, 2nd Edition 地球化学经典教科书第二版 300
Idoxuridine 260
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 3196852
求助须知:如何正确求助?哪些是违规求助? 2845520
关于积分的说明 8054339
捐赠科研通 2510135
什么是DOI,文献DOI怎么找? 1342314
科研通“疑难数据库(出版商)”最低求助积分说明 639355
邀请新用户注册赠送积分活动 608669