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
Breathe完成签到 ,获得积分10
刚刚
wandong应助kkhenry采纳,获得10
刚刚
oyl完成签到,获得积分10
刚刚
刚刚
roc221发布了新的文献求助30
1秒前
木子青山完成签到,获得积分10
1秒前
Jasper应助ning采纳,获得10
2秒前
不忘初心完成签到,获得积分10
2秒前
我是老大应助misalia采纳,获得10
2秒前
科研通AI2S应助lou采纳,获得10
2秒前
3秒前
实验农民工完成签到,获得积分10
3秒前
Jerryya完成签到,获得积分10
3秒前
4秒前
林珏发布了新的文献求助10
4秒前
大糖糕僧完成签到 ,获得积分10
4秒前
4秒前
5秒前
英姑应助科研通管家采纳,获得10
5秒前
科研通AI2S应助科研通管家采纳,获得10
5秒前
英俊的铭应助科研通管家采纳,获得10
5秒前
SciGPT应助科研通管家采纳,获得10
6秒前
斯文败类应助科研通管家采纳,获得10
6秒前
FashionBoy应助科研通管家采纳,获得10
6秒前
天天快乐应助科研通管家采纳,获得10
6秒前
wanci应助科研通管家采纳,获得10
6秒前
古德猫宁完成签到 ,获得积分10
6秒前
无花果应助科研通管家采纳,获得30
6秒前
小蘑菇应助科研通管家采纳,获得30
6秒前
科研通AI2S应助科研通管家采纳,获得10
6秒前
6秒前
6秒前
zoe发布了新的文献求助10
6秒前
丘比特应助科研通管家采纳,获得10
6秒前
完美世界应助矮小的睫毛采纳,获得10
6秒前
情怀应助科研通管家采纳,获得10
6秒前
Ava应助科研通管家采纳,获得30
7秒前
7秒前
不配.应助科研通管家采纳,获得10
7秒前
小马甲应助S1008采纳,获得10
7秒前
高分求助中
Spray / Wall-interaction Modelling by Dimensionless Data Analysis 2000
Femoral insertion of the ACL. Radiographic quadrant method 1000
ALA生合成不全マウスでの糖代謝異常の分子機構解析 520
Mathematics and Finite Element Discretizations of Incompressible Navier—Stokes Flows 500
2024 Medicinal Chemistry Reviews 400
Dictionary of socialism 350
Geochemistry, 2nd Edition 地球化学经典教科书第二版 300
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 3194457
求助须知:如何正确求助?哪些是违规求助? 2843351
关于积分的说明 8044600
捐赠科研通 2507836
什么是DOI,文献DOI怎么找? 1340139
科研通“疑难数据库(出版商)”最低求助积分说明 638898
邀请新用户注册赠送积分活动 607764