爪蟾卵母细胞跨膜转运功能分析服务

为什么用爪蟾卵母细胞研究转运蛋白？Why use Xenopus oocytes for transporter research?

爪蟾卵母细胞内源性转运体背景极低，对外源注射的 cRNA 翻译效率高，能在质膜上大量表达目的转运蛋白；细胞体积大（约 1 µL）使精准底物摄取检测成为可能。这一系统已被全球数百个实验室用于研究氨基酸、糖、核苷酸、有机酸、金属离子、农药及药物等各类底物的跨膜转运。Xenopus oocytes have very low endogenous transporter background and efficiently translate injected cRNA to produce large amounts of the target transporter on the plasma membrane. Their large volume (~1 µL) enables precise substrate uptake measurements. This system has been used by hundreds of labs worldwide to study transmembrane transport of amino acids, sugars, nucleotides, organic acids, metal ions, pesticides and drugs.

植物转运体：HKT（Na⁺/K⁺）、MATE、ABC、NRT、MTP、铁转运、硅酸转运等Plant transporters: HKT (Na⁺/K⁺), MATE, ABC, NRT, MTP, iron, silicate
动物转运体：氨基酸转运体（LAT、SNAT）、糖转运体（GLUT、SGLT）、有机酸转运体Animal transporters: amino acid (LAT, SNAT), glucose (GLUT, SGLT), organic acid
微生物转运体：细菌离子通道、真菌营养转运体功能鉴定Microbial transporters: bacterial ion channels, fungal nutrient transporter characterization

两种互补检测方法Two complementary detection methods

¹³C 同位素示踪 + 荧光探针，双轨验证¹³C isotope tracing + fluorescent probes — dual verification

方法 01

¹³C 稳定同位素示踪法（LC-MS 定量）¹³C stable-isotope tracing (LC-MS quantification)

在摄取实验中加入含 ¹³C 标记的底物（如 ¹³C₆-葡萄糖、¹³C₅-谷氨酸等），结束摄取后立即将卵母细胞裂解，通过液相色谱 – 质谱联用（LC-MS/MS）对胞内 ¹³C 标记底物进行定量。由于天然背景物质为 ¹²C，¹³C 标记物信号特异性极高，可精准区分转运摄取与非特异性结合。¹³C-labeled substrates (e.g., ¹³C₆-glucose, ¹³C₅-glutamate) are added to the uptake solution. After the uptake period, oocytes are immediately homogenized and ¹³C-labeled intracellular substrate is quantified by LC-MS/MS. Since background molecules are ¹²C, the ¹³C signal is highly specific, accurately distinguishing transporter-mediated uptake from non-specific binding.

优势Advantages

绝对定量，可计算 V_max（pmol / oocyte / min）Absolute quantification, V_max in pmol/oocyte/min
高特异性，背景极低High specificity, minimal background
适用于所有可同位素标记的底物Applicable to all isotopically labelable substrates
可同时检测多种底物竞争性摄取Simultaneous detection of multi-substrate competition

方法 02

荧光探针实时动力学分析Fluorescent probe real-time kinetics

利用特异性荧光底物（如葡萄糖探针 2-NBDG、氨基酸类似物、金属离子螯合荧光指示剂 Fluo-4 等）通过共聚焦显微镜或荧光酶标仪实时监测卵母细胞胞内荧光强度变化，计算初速率与底物浓度关系（Michaelis-Menten 方程）。特别适合转运体抑制剂的快速高通量筛选。Fluorescent substrates (e.g., glucose analogue 2-NBDG, amino acid fluorescent analogues, metal-ion chelating dyes such as Fluo-4) are monitored in real time via confocal microscopy or fluorescence plate reader. Initial rates at multiple substrate concentrations are fitted to the Michaelis-Menten equation. Particularly suited for high-throughput inhibitor screening.

优势Advantages

实时动态监测，时间分辨率高Real-time monitoring, high temporal resolution
无需 LC-MS 设备，通量更高No LC-MS required, higher throughput
适合快速 IC₅₀ 筛选Ideal for rapid IC₅₀ screening
可与共聚焦成像联合，同步观察转运蛋白定位Combinable with confocal imaging for simultaneous localization

服务流程Workflow

从目的基因到转运动力学报告From target gene to transport kinetics report

1

目的基因构建与 cRNA 体外转录Gene construction & cRNA in-vitro transcription

2

卵母细胞显微注射（50 nL / 个）Oocyte microinjection (50 nL/cell)

3

ND96 孵育 1–3 天（18–22℃）ND96 incubation 1–3 days (18–22 °C)

4

¹³C 底物摄取实验 / 荧光动力学检测¹³C substrate uptake / fluorescence kinetics

5

LC-MS 定量 / 荧光分析 → K_m、V_max、IC₅₀ 报告LC-MS / fluorescence analysis → K_m, V_max, IC₅₀ report

项目Item	¹³C LC-MS 法	荧光探针法Fluorescent probe
定量方式Quantification	绝对定量（pmol/cell）Absolute (pmol/cell)	相对荧光强度（ΔF）Relative ΔF
通量Throughput	中（单批次 10–20 条件）Medium (10–20 conditions/batch)	高（适合药物筛选）High (suitable for screening)
底物要求Substrate requirement	可 ¹³C 标记（常见代谢物）Isotopically labelable	有荧光探针或类似物Fluorescent probe/analogue available
客户提供Client provides	基因序列 / 质粒 / cRNA；底物（如需）；抑制剂（如需）Gene / plasmid / cRNA; substrate (if needed); inhibitors (if needed)
交付物Deliverables	完整技术报告 + 原始数据 + K_m/V_max/IC₅₀ 图表Full report + raw data + K_m/V_max/IC₅₀ figures

精准鉴定你的转运蛋白Precisely characterize your transporter

提供目的基因序列，我们制备 cRNA、完成转运检测并计算动力学参数，出具专业分析报告。Submit your gene sequence — we prepare cRNA, run the transport assay, calculate kinetic parameters and deliver a professional report.

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