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免疫熒光的高通量細(xì)胞蛋白質(zhì)檢測技術(shù)是怎么樣的？

更新時間：2026-06-09 點(diǎn)擊次數(shù)：22

免疫熒光的高通量細(xì)胞蛋白質(zhì)檢測技術(shù)是一種定量免疫熒光測定，在多孔板（優(yōu)化為96孔或384孔格式）中進(jìn)行，結(jié)合了Western blotting的特異性與ELISA的可重復(fù)性和通量。

該測定也稱為細(xì)胞印跡、細(xì)胞基ELISA、細(xì)胞內(nèi)ELISA（ICE）和FACE（快速活化細(xì)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(xì)胞中的蛋白質(zhì)®
使用光譜上不同的熒光染料共軛物定量多個靶點(diǎn)
快速且準(zhǔn)確地測量多個樣本中的相對蛋白質(zhì)水平
在相關(guān)細(xì)胞環(huán)境中檢測蛋白質(zhì)
歸一化為細(xì)胞數(shù)，便于準(zhǔn)確定量和比較各孔間蛋白質(zhì)表達(dá)

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(tǒng)的工作流程自動化了分析的關(guān)鍵步驟。對于In-Cell Western Assays，可以創(chuàng)建自定義模板或使用以下六個預(yù)設(shè)模板之一：®

細(xì)胞染色線性性：確保歸一化方法和靶材的信號強(qiáng)度均在其線性范圍內(nèi)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(shí)驗(yàn)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(shí)驗(yàn)的最佳固定和滲透條件
Z′因子測定：測試測定的質(zhì)量和穩(wěn)健性
靶點(diǎn)分析：量化治療或病癥對靶點(diǎn)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(zhì)磷酸化
（藥物化合物對信號通路的影響;信號傳導(dǎo)的時序/動力學(xué); IC50determination)
GPCR激活
Tau蛋白的積累與抑制
基因表達(dá)水平
病毒滴度/載量
凋亡：Caspase-3激活
細(xì)胞表面蛋白與受體內(nèi)化
RNAi療效
細(xì)胞增殖檢測

In-Cell Western Assay是如何工作的？

本測定基于標(biāo)準(zhǔn)免疫熒光方法。

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

In-Cell Western Assay可重復(fù)且精確

本測定比Western Blotting具有更高的復(fù)現(xiàn)性和精度。細(xì)胞內(nèi)西方測定顯示以下特征：

更短的協(xié)議
顯著更小的標(biāo)準(zhǔn)差
復(fù)現(xiàn)變異系數(shù)極低的測量值（CV）（見圖1）
可以輕松運(yùn)行多次復(fù)制以提高準(zhǔn)確性（見圖2）
表征廣泛的細(xì)胞信號傳導(dǎo)參數(shù)
與西部片相比，信號的增減特征非常相似
在優(yōu)化條件下和實(shí)驗(yàn)設(shè)計(jì)下，能夠產(chǎn)生優(yōu)異的Z′因子

Z′因子是統(tǒng)計(jì)效應(yīng)大小的衡量指標(biāo)，可用于評估檢測反應(yīng)是否需要進(jìn)一步調(diào)查。Z′因子可以為檢測的可重復(fù)性提供一定的指示。Z′ 是通過運(yùn)行大量正對照和陰對照，并確定正負(fù)對照之間的間隔來計(jì)算的。如果由于對照組內(nèi)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(yōu)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(xiàng)研究比較了本測定法和WB 測量磷酸化肌球蛋白調(diào)節(jié)輕鏈（PMLC20）。1使用催產(chǎn)素刺激的子宮肌細(xì)胞初級培養(yǎng)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(jié)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(guān)性

本測定法和WB結(jié)果及其他檢測結(jié)果高度相關(guān)。

In-Cell Western Assay與Western Blotting的相關(guān)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(qiáng)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(shù)值與敏感度
改進(jìn)的復(fù)制性和精度1

顯著更小的標(biāo)準(zhǔn)差
用非常低的CV復(fù)現(xiàn)測量結(jié)果

更高的通量，便于并行處理大量樣品或復(fù)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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