mRNA / cRNA 注射及卵母细胞蛋白表达mRNA/cRNA injection & oocyte protein expression
向爪蟾卵母细胞显微注射 mRNA / cRNA，实现离子通道、转运蛋白、受体及膜蛋白的异源表达；配合 双电极电压钳（TEVC）功能检测、FLAG / GFP 标签融合表达、Western Blot 蛋白定量及激光共聚焦显微镜膜定位验证，提供从分子到功能的全面数据包。Microinjection of mRNA/cRNA into Xenopus oocytes for heterologous expression of ion channels, transporters, receptors and membrane proteins — combined with TEVC functional recording, FLAG/GFP-tag fusion expression, Western Blot quantification and confocal membrane-localization imaging for a complete molecular-to-function data package.
为什么用爪蟾卵母细胞？Why Xenopus oocytes?
爪蟾卵母细胞体积大、内源通道背景低、翻译与翻译后修饰机制完善，是异源表达膜蛋白并进行电生理 / 转运功能研究的经典系统。Large, with low endogenous channel background and full translational machinery, Xenopus oocytes are the classic system for heterologous membrane-protein expression and electrophysiology.
膜蛋白验证：FLAG/GFP 标签融合，Western Blot 定量，共聚焦确认质膜定位Membrane protein verification: FLAG/GFP fusion, Western Blot quantification, confocal plasma-membrane localization
膜蛋白表达验证Membrane protein verification
FLAG / GFP 标签 · Western Blot · 激光共聚焦显微镜FLAG/GFP tag · Western Blot · Confocal microscopy
在完成电生理或转运功能检测的同时，中心提供分子标签融合表达与多模态验证服务，从蛋白表达量和亚细胞定位两个维度确认目的蛋白正确插入质膜，为功能数据提供分子层面佐证。Alongside electrophysiology or transport assays, we provide molecular-tag fusion expression with multi-modal verification — quantifying protein expression and confirming plasma-membrane localization to support your functional data at the molecular level.
01 · 标签构建Tag construction
FLAG / GFP / HA / mCherry 融合表达FLAG / GFP / HA / mCherry fusion expression
在目的蛋白 N 端或 C 端插入标签序列（客户自建或中心协助克隆），体外转录获得带标签 cRNA，注射卵母细胞后 1–3 天检测。GFP / mCherry 可直接荧光成像；FLAG / HA 可用于免疫染色、Co-IP 等。Tag sequences are inserted at the N- or C-terminus (client-built or center-cloned), transcribed to tagged cRNA and injected. GFP/mCherry enable direct live-cell imaging; FLAG/HA enable immunostaining and Co-IP.
02 · Western Blot
蛋白表达量与修饰状态检测Protein expression level & modification status
注射后按时间点收集卵母细胞，裂解提取总蛋白或质膜蛋白组分，SDS-PAGE 分离后用抗 FLAG / GFP / HA 抗体或目的蛋白特异抗体（客户提供）进行 Western Blot 检测。评估表达量、分子量及糖基化等翻译后修饰，以水注射对照组排除内源蛋白干扰。Oocytes are harvested at defined time-points; total or plasma-membrane protein fractions are resolved by SDS-PAGE and detected with anti-FLAG/GFP/HA antibodies or client-supplied target-specific antibodies. This evaluates expression level, molecular weight and glycosylation, compared against water-injected controls.
03 · 共聚焦成像Confocal imaging
质膜定位实时可视化Real-time plasma-membrane localization
对注射了 GFP / mCherry 融合 cRNA 的卵母细胞进行激光共聚焦成像，活细胞状态下直接观察蛋白亚细胞定位。正确定位于质膜的蛋白呈现清晰的质膜环状荧光；内质网或内体滞留则提示需优化构建体。可配合 DiI / DiO 膜染料或凝集素（Lectin）进行共定位分析，确认质膜归属。Live-cell laser confocal imaging of oocytes expressing GFP/mCherry-fused proteins directly visualizes subcellular localization. Correct plasma-membrane targeting appears as a distinct fluorescent ring at the cell periphery; ER or endosome retention indicates construct optimization is needed. Co-localization with DiI/DiO dyes or lectin confirms plasma-membrane identity.
验证方法Method
检测指标Readout
所需标签 / 抗体Required tag / antibody
交付物Deliverables
Western Blot
蛋白分子量与表达量MW & expression level
FLAG / GFP / HA（或客户自备抗体）
凝胶图 + 定量图表Gel image + quantification
激光共聚焦Confocal
质膜 / 细胞器定位PM / organelle localization
GFP / mCherry / YFP
高分辨率荧光图像 + Z 轴扫描High-res images + Z-stack
质膜蛋白分级Membrane fractionation
质膜蛋白富集确认PM enrichment
任意标签或自备抗体Any tag or custom antibody
分级 WB + 比较数据Fractionation WB + comparison
服务流程Workflow
从 cRNA 到多维度数据From cRNA to multi-dimensional data
1
基因克隆与 cRNA 体外转录（可含 FLAG / GFP 标签）Gene cloning & cRNA synthesis (with optional FLAG/GFP)
2
卵母细胞显微注射Oocyte microinjection
3
表达孵育（1–4 天）Expression incubation (1–4 d)
4
表达验证（Western Blot / 共聚焦定位）Expression verification (Western / confocal)
