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免疫荧光的高通量细胞蛋白质检测技术是怎么样的？

更新时间：2026-06-09 点击次数：31

免疫荧光的高通量细胞蛋白质检测技术是一种定量免疫荧光测定，在多孔板（优化为96孔或384孔格式）中进行，结合了Western blotting的特异性与ELISA的可重复性和通量。

该测定也称为细胞印迹、细胞基ELISA、细胞内ELISA（ICE）和FACE（快速活化细胞基ELISA）。通过In-Cell Western，可以：

利用靶特异性一级抗体和IRDye二级抗体检测固定和渗透细胞中的蛋白质®
使用光谱上不同的荧光染料共轭物定量多个靶点
快速且准确地测量多个样本中的相对蛋白质水平
在相关细胞环境中检测蛋白质
归一化为细胞数，便于准确定量和比较各孔间蛋白质表达

Empiria Studio 软件为多种多孔板式测定提供分步工作流程和板式模板，包括In-Cell Western Assay。Empiria Studio 中这些简单且系统的工作流程自动化了分析的关键步骤。对于In-Cell Western Assays，可以创建自定义模板或使用以下六个预设模板之一：®

细胞染色线性性：确保归一化方法和靶材的信号强度均在其线性范围内被检测到
抗体滴定：确定提供最佳信号和*低背景的抗体浓度
阻断剂评估：确定实验中最佳阻断缓冲液或阻断缓冲剂与抗体组合
固定与渗透评估：确定实验的最佳固定和渗透条件
Z′因子测定：测试测定的质量和稳健性
靶点分析：量化治疗或病症对靶点的影响

In-Cell Western Assay与Western Blotting工作流程比较

In-Cell Western Assay已被用于分析：

蛋白质磷酸化
（药物化合物对信号通路的影响;信号传导的时序/动力学; IC50determination)
GPCR激活
Tau蛋白的积累与抑制
基因表达水平
病毒滴度/载量
凋亡：Caspase-3激活
细胞表面蛋白与受体内化
RNAi疗效
细胞增殖检测

In-Cell Western Assay是如何工作的？

本测定基于标准免疫荧光方法。

多孔板中的种子细胞
细胞处理（可选）
固定单元
细胞渗透化
阻断单元
制备初级抗体溶液
培养一级抗体溶液
洗盘
准备二级抗体溶液和细胞染色剂，如CellTag™ 520染色剂或CellTag 700染色剂
培养二级抗体溶液和细胞染色
洗盘
图像多孔板

In-Cell Western Assay可重复且精确

本测定比Western Blotting具有更高的复现性和精度。细胞内西方测定显示以下特征：

更短的协议
显著更小的标准差
复现变异系数极低的测量值（CV）（见图1）
可以轻松运行多次复制以提高准确性（见图2）
表征广泛的细胞信号传导参数
与西部片相比，信号的增减特征非常相似
在优化条件下和实验设计下，能够产生优异的Z′因子

Z′因子是统计效应大小的衡量指标，可用于评估检测反应是否需要进一步调查。Z′因子可以为检测的可重复性提供一定的指示。Z′ 是通过运行大量正对照和阴对照，并确定正负对照之间的间隔来计算的。如果由于对照组内部差异很大，它们重叠或几乎重叠（Z′ < 0.5），该检测法对筛选无效。优异的检测方法显示阳性与阴性对照的良好分离（Z′ > 0.5）。 2,4,5

2010年一项研究比较了本测定法和WB 测量磷酸化肌球蛋白调节轻链（PMLC20）。1使用催产素刺激的子宫肌细胞初级培养物，评估两种磷酸分析方法的特异性、敏感性和精度。

本测定法和WB分析结果非常相似（见图1）。本测定提供了更好的精度、更低的变异性和更小的CV。

Figure 1. Intra-assay variability of Western blots and In-Cell Western Assays. A) GAPDH and PMLC20 were detected on duplicate Western blots (13 replicates per blot, not shown). Band intensities are expressed as a proportion of the mean of the thirteen samples on each blot (blot 1 in blue, blot 2 in red). CV was 0.27. B) Signal intensities from In-Cell Western wells are plotted as a proportion of the mean values for each of two plates (plate 1 in blue, plate 2 in red). CV ranged from 0.08 to 0.16.1

Figure 2. Replicability of In-Cell Western Assay for siRNA screening via a correlation plot. A pilot small molecule siRNA screen was performed for inhibitors of mTORC1 signaling. A library of ~2500 known bioactive compounds was used. Compounds with average Z-score < -2 are considered hits (red circles). Known inhibitors of mTORC1 signaling found in the library are shown in green. Plate-to-plate consistency is shown for a representative plate from the library. R2 = 0.91, indicating exceptional plate-to-plate reproducibility. Adapted with permission from Hoffman, GR et al.2

与其他测定的相关性

本测定法和WB结果及其他检测结果高度相关。

In-Cell Western Assay与Western Blotting的相关性

In-Cell Western Assay可用于筛选抑制剂以确定IC50或用于确定EC的增强或刺激因素50.与Western Blotting相比，In-Cell Western Assay显示：

信号的增减曲线非常相似，IC也相似50数值与敏感度
改进的复制性和精度1

显著更小的标准差
用非常低的CV复现测量结果

更高的通量，便于并行处理大量样品或复制品

In Figure 3 and Figure 4, TPA dose response was assessed by In-Cell Western Assay and Western blot. In-Cell Western EC50 values were calculated (Figure 3) that closely match the EC50 values calculated in a Western blot analog (Figure 4).8

Figure 3. EC50 values were calculated that match expected results. A) The multiwell plate is shown for this In-Cell Western Assay with the amount of TPA used to treat wells shown in nM. Wells in the same column are technical replicates. B) In this chart, pan-ERK1 signal is normalized to CellTag 520 Stain, and the fold change for pan-ERK1 treatments are calculated with the 0 nM treatment as the denominator. The 0 nM treatment is set as the Control on the Set up charts page in the Empiria Studio Software Target Analysis workflow. C) p-ERK1/2 signals are normalized to CellTag 520 Stain. The fold change is calculated the same way as described in part A. The EC50 value is 2.6nM (calculated outside of Empiria Studio Software). D) p-ERK1/2 signals are normalized to panERK1. Fold change is calculated as described in part A. The EC50 value is 2.7 nM (calculated outside of Empiria Studio Software).

Figure 4. EC50 values were calculated that match expected results. A) Western blot images for this assay are shown. The 520 nm channel with Revert™ 520 Total Protein Stain is shown on the left, and the 700 and 800 nm channels with pan-ERK1 and p-ERK1/2 bands are shown overlaid on the right. B) In this chart, pan-ERK1 signal is normalized to Revert 520 Total Protein Stain, and the fold change for pan-ERK1 treatments are calculated with the 0 nM treatment as the denominator. The 0 nM treatment is set as the Control on the Set up charts page in the Empiria Studio Software Western blot Target Analysis workflow. C) p-ERK1 signals are normalized to Revert 520 Total Protein Stain. The fold change for p-ERK1 is calculated using the same method described in part B for pan-ERK1. The EC50 value is 4.3nM (calculated outside of Empiria Studio Software). D) p-ERK2 signals are normalized to Revert 520 Total Protein Stain. The fold change for p-ERK2 is calculated using the same method described in part B for pan-ERK1. The EC50 value is 3.9 nM (calculated outside of Empiria Studio Software).

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