Looking for step-by-step instructions to develop your assay? Get your free copy of the In-Cell Western Assay Development Handbook.
DownloadThe In-Cell Western Assay is a quantitative immunofluorescence assay performed in multiwell plates (optimized for 96- or 384-well format) that combines the specificity of Western blotting with the replicability and throughput of ELISA.
In-Cell Western Assays are also called cytoblots, cell-based ELISA, In-Cell ELISA (ICE), and FACE (Fast Activated Cell-based ELISA). With In-Cell Western Assays, you can:
Empiria Studio® Software has step-by-step workflows and Plate Templates for various types of multiwell plate assays, including the In-Cell Western Assay. These straightforward and systematic workflows in Empiria Studio automate the critical steps of your analysis. For In-Cell Western Assays, create a custom template or use one of the six preset templates for:
In-Cell Western Assays have been used for analysis of:
The In-Cell Western Assay is based on standard immunofluorescent methods.
In-Cell Western Assays provide greater replicability and precision than Western blots. In-Cell Western Assays exhibit the following characteristics:
Z′-Factor is a measure of statistical effect size and can be used to assess if a response to an assay requires further investigation. Z′-Factor can provide some indication of the replicability of an assay. Z′ is calculated by running a large number of positive and negative controls and determining how much separation there is between positive and negative controls. If they overlap, or nearly overlap (Z′ < 0.5), due to large amounts of variation within the controls, the assay is useless for screening. An excellent assay exhibits good separation between positive and negative controls (Z′ > 0.5).2, 4, 5
A 2010 study compared In-Cell Western Assays and Western blotting (WB) for measurement of phosphorylated myosin regulatory light chain (PMLC20).1 Primary cultures of uterine myocytes stimulated with oxytocin were used to assess specificity, sensitivity, and precision of the two methods for phospho-analysis.
In-Cell Western Assay and Western blot analysis yielded very similar results (Figure 1). In-Cell Western Assays offered superior precision, reduced variability, and smaller CVs.
In-Cell Western Assay results correlate well with immunoblot results and other assays.
The In-Cell Western Assay can be used to screen inhibitors for determining IC50 or enhancement or stimulation factors for determining EC50. Compared to Western blotting, In-Cell Western Assays display:
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
GPCR activity assays primarily monitor upstream signaling events, such as accumulation of cyclic AMP (cAMP). Downstream events, such as phosphorylation of cAMP response element binding protein (CREB), may also be useful readouts.3, 6
The In-Cell Western method is commonly used to assess IC50 and has been shown to produce comparable IC50 results to other assays, such as radioligand binding affinities and cAMP accumulation assays.3
| Method of IC50 Determination | Result |
|---|---|
| CREB In-Cell Western (pKB) | 8.72±0.13 |
| cAMP ELISA (pKB) | 8.72±0.07 |
| Radioligand binding assay (pKi) | 8.40±0.05 |
Normalization makes In-Cell Western analysis more precise by correcting for well-to-well variation in cell number.
Choose the normalization approach that best suits your experiment.
Post-translational modification (PTM) normalizes to a specific modification of a target protein against all target protein, regardless of modification. The target protein is used as its own internal control.
PTM normalization employs two primary antibodies raised in different hosts:
To simultaneously detect multiple target proteins in the In-Cell Western Assay, you must use secondary antibodies labeled with different fluorophores (e.g., IRDye 680RD Secondary Antibodies for 700 nm channel and IRDye 800CW Secondary Antibodies for 800 nm channel).
It may be possible to use the target protein as its own internal control for detection of a phospho-protein.
Cell number normalization is a fast and inexpensive approach because no additional antibodies are required. Options include staining with CellTag 700 Stain or CellTag 520 Stain.
CellTag 700 Stain and CellTag 520 Stain are near-infrared fluorescent cell stains that provide accurate estimation of cell number for In-Cell Western Assay applications. The stain accumulates in both the nucleus and the cytoplasm of permeabilized cells and provides linear fluorescent signal across a wide range of cell types and cell membranes. This method is fast and easy because staining is combined with secondary antibody incubation.
CellTag stains are ideal internal controls for most In-Cell Western Assays because of their broad linear ranges and need for minimal validation. For example, CellTag 700 Stain is ideal for 2-color In-Cell Western Assays, in which the protein of interest is detected in the 800 nm channel and the internal control in the 700 nm channel. If your imager can capture visible fluorescence, such as the Odyssey® M Imager, then CellTag 520 Stain is ideal because it helps detect two proteins of interest in the 700 and 800 nm channels and the internal control in the 520 nm channel.
Using this method, cells are covalently labeled using cellular lysine residues on cellular proteins with IRDye 800CW Infrared Dyes.7 Cell labeling is an inexpensive method with high sensitivity and a broad linear range (~200 to 200,000 cells/well), and results are unaffected by changes in nuclear DNA. Cell labeling with reactive dye is ideal for On-Cell Western assays because the cells are not permeabilized.
See some published examples of In-Cell Western Assays
View publications