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免疫熒光的高通量細胞蛋白質檢測技術是怎么樣的？

更新時間：2026-06-09 點擊次數：164

免疫熒光的高通量細胞蛋白質檢測技術是一種定量免疫熒光測定，在多孔板（優化為96孔或384孔格式）中進行，結合了Western blotting的特異性與ELISA的可重復性和通量。

該測定也稱為細胞印跡、細胞基ELISA、細胞內ELISA（ICE）和FACE（快速活化細胞基ELISA）。通過In-Cell Western，可以：

利用靶特異性一級抗體和IRDye二級抗體檢測固定和滲透細胞中的蛋白質®
使用光譜上不同的熒光染料共軛物定量多個靶點
快速且準確地測量多個樣本中的相對蛋白質水平
在相關細胞環境中檢測蛋白質
歸一化為細胞數，便于準確定量和比較各孔間蛋白質表達

Empiria Studio 軟件為多種多孔板式測定提供分步工作流程和板式模板，包括In-Cell Western Assay。Empiria Studio 中這些簡單且系統的工作流程自動化了分析的關鍵步驟。對于In-Cell Western Assays，可以創建自定義模板或使用以下六個預設模板之一：®

細胞染色線性性：確保歸一化方法和靶材的信號強度均在其線性范圍內被檢測到
抗體滴定：確定提供最佳信號和*低背景的抗體濃度
阻斷劑評估：確定實驗中最佳阻斷緩沖液或阻斷緩沖劑與抗體組合
固定與滲透評估：確定實驗的最佳固定和滲透條件
Z′因子測定：測試測定的質量和穩健性
靶點分析：量化治療或病癥對靶點的影響

In-Cell Western Assay與Western Blotting工作流程比較

In-Cell Western Assay已被用于分析：

蛋白質磷酸化
（藥物化合物對信號通路的影響;信號傳導的時序/動力學; IC50determination)
GPCR激活
Tau蛋白的積累與抑制
基因表達水平
病毒滴度/載量
凋亡：Caspase-3激活
細胞表面蛋白與受體內化
RNAi療效
細胞增殖檢測

In-Cell Western Assay是如何工作的？

本測定基于標準免疫熒光方法。

多孔板中的種子細胞
細胞處理（可選）
固定單元
細胞滲透化
阻斷單元
制備初級抗體溶液
培養一級抗體溶液
洗盤
準備二級抗體溶液和細胞染色劑，如CellTag™ 520染色劑或CellTag 700染色劑
培養二級抗體溶液和細胞染色
洗盤
圖像多孔板

In-Cell Western Assay可重復且精確

本測定比Western Blotting具有更高的復現性和精度。細胞內西方測定顯示以下特征：

更短的協議
顯著更小的標準差
復現變異系數極低的測量值（CV）（見圖1）
可以輕松運行多次復制以提高準確性（見圖2）
表征廣泛的細胞信號傳導參數
與西部片相比，信號的增減特征非常相似
在優化條件下和實驗設計下，能夠產生優異的Z′因子

Z′因子是統計效應大小的衡量指標，可用于評估檢測反應是否需要進一步調查。Z′因子可以為檢測的可重復性提供一定的指示。Z′ 是通過運行大量正對照和陰對照，并確定正負對照之間的間隔來計算的。如果由于對照組內部差異很大，它們重疊或幾乎重疊（Z′ < 0.5），該檢測法對篩選無效。優異的檢測方法顯示陽性與陰性對照的良好分離（Z′ > 0.5）。 2,4,5

2010年一項研究比較了本測定法和WB 測量磷酸化肌球蛋白調節輕鏈（PMLC20）。1使用催產素刺激的子宮肌細胞初級培養物，評估兩種磷酸分析方法的特異性、敏感性和精度。

本測定法和WB分析結果非常相似（見圖1）。本測定提供了更好的精度、更低的變異性和更小的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

與其他測定的相關性

本測定法和WB結果及其他檢測結果高度相關。

In-Cell Western Assay與Western Blotting的相關性

In-Cell Western Assay可用于篩選抑制劑以確定IC50或用于確定EC的增強或刺激因素50.與Western Blotting相比，In-Cell Western Assay顯示：

信號的增減曲線非常相似，IC也相似50數值與敏感度
改進的復制性和精度1

顯著更小的標準差
用非常低的CV復現測量結果

更高的通量，便于并行處理大量樣品或復制品

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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