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
斯文的凝珍完成签到,获得积分10
4秒前
酷波er应助zzx采纳,获得10
8秒前
科研通AI2S应助科研通管家采纳,获得10
14秒前
19秒前
CHINA_C13发布了新的文献求助30
25秒前
充电宝应助xulin采纳,获得10
28秒前
Benjamin完成签到 ,获得积分10
31秒前
Da完成签到,获得积分10
33秒前
随机子应助CHINA_C13采纳,获得30
34秒前
38秒前
xulin完成签到,获得积分10
38秒前
38秒前
李爱国应助颜哈哈采纳,获得30
39秒前
struggling2026完成签到 ,获得积分10
40秒前
YSY完成签到,获得积分10
45秒前
49秒前
57秒前
我像风一样自由完成签到 ,获得积分10
58秒前
shit完成签到 ,获得积分10
59秒前
1分钟前
hhan完成签到,获得积分10
1分钟前
buzhinianjiu完成签到 ,获得积分10
1分钟前
hhan发布了新的文献求助10
1分钟前
1分钟前
DYYYYYY发布了新的文献求助10
1分钟前
1分钟前
1分钟前
香蕉觅云应助DYYYYYY采纳,获得10
1分钟前
颜哈哈发布了新的文献求助30
1分钟前
第二十篇完成签到,获得积分10
1分钟前
xiubo128完成签到,获得积分10
1分钟前
激情的晓凡完成签到,获得积分10
1分钟前
颜哈哈完成签到,获得积分10
1分钟前
xiubo128完成签到,获得积分10
1分钟前
LIGNET完成签到,获得积分10
1分钟前
1分钟前
科研通AI2S应助曾经易真采纳,获得10
1分钟前
1347365881应助jiang采纳,获得10
1分钟前
1分钟前
1分钟前
高分求助中
Evolution 3rd edition 1500
保险藏宝图 1000
Lire en communiste 1000
Mantiden: Faszinierende Lauerjäger Faszinierende Lauerjäger 700
PraxisRatgeber: Mantiden: Faszinierende Lauerjäger 700
Mathematics and Finite Element Discretizations of Incompressible Navier—Stokes Flows 500
A new species of Coccus (Homoptera: Coccoidea) from Malawi 500
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 3183637
求助须知:如何正确求助?哪些是违规求助? 2833619
关于积分的说明 7995220
捐赠科研通 2495932
什么是DOI,文献DOI怎么找? 1331799
科研通“疑难数据库(出版商)”最低求助积分说明 636425
邀请新用户注册赠送积分活动 603582