亲爱的研友该休息了!由于当前在线用户较少,发布求助请尽量完整的填写文献信息,科研通机器人24小时在线,伴您度过漫漫科研夜!身体可是革命的本钱,早点休息,好梦!

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
2秒前
八风乱动发布了新的文献求助10
7秒前
18秒前
八风乱动发布了新的文献求助10
25秒前
34秒前
研友_VZG7GZ应助科研通管家采纳,获得10
36秒前
八风乱动发布了新的文献求助10
39秒前
TXZ06完成签到,获得积分10
46秒前
55秒前
八风乱动发布了新的文献求助10
1分钟前
1分钟前
八风乱动发布了新的文献求助10
1分钟前
1分钟前
1分钟前
cacaldon发布了新的文献求助20
1分钟前
八风乱动完成签到,获得积分10
1分钟前
Raunio发布了新的文献求助10
1分钟前
cacaldon完成签到,获得积分10
1分钟前
YifanWang应助andrele采纳,获得10
2分钟前
2分钟前
搞怪熊猫发布了新的文献求助10
2分钟前
2分钟前
搞怪熊猫完成签到,获得积分10
3分钟前
3分钟前
3分钟前
ShowMaker应助kakarot48848141采纳,获得20
4分钟前
kakarot48848141完成签到,获得积分20
4分钟前
汉堡包应助piglet采纳,获得10
4分钟前
充电宝应助科研通管家采纳,获得10
4分钟前
4分钟前
amengptsd完成签到,获得积分10
5分钟前
啊啊啊发布了新的文献求助10
5分钟前
glacier发布了新的文献求助10
5分钟前
6分钟前
巅峰囚冰完成签到,获得积分10
6分钟前
Lucas应助科研通管家采纳,获得10
6分钟前
成就丸子完成签到 ,获得积分10
6分钟前
draven007发布了新的文献求助10
6分钟前
在水一方应助老实铁身采纳,获得30
6分钟前
Lucas应助draven007采纳,获得10
6分钟前
高分求助中
Spray / Wall-interaction Modelling by Dimensionless Data Analysis 2000
Write Like a Chemist: A Guide and Resource (第二版) 600
Mixed-anion Compounds 600
A new species of Velataspis (Hemiptera Coccoidea Diaspididae) from tea in Assam 500
Earth System Geophysics 500
Aspects of Babylonian celestial divination: the lunar eclipse tablets of Enūma Anu Enlil 500
Geochemistry, 2nd Edition 地球化学经典教科书第二版 401
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 3200763
求助须知:如何正确求助?哪些是违规求助? 2850509
关于积分的说明 8072174
捐赠科研通 2514255
什么是DOI,文献DOI怎么找? 1346981
科研通“疑难数据库(出版商)”最低求助积分说明 640305
邀请新用户注册赠送积分活动 610421