New Poster on ICW Assay

A Functional siRNA Screen for Novel Regulators of mTORC1 Signaling by Greg Hoffman, Max Hsia, and John Blenis--Harvard Medical School, Dept. of Cell Biology. Download a PDF of the poster here.

Microplate Setup Guide

Construct an In-Cell Western experimental plan with this easy-to-follow guide. Download the PDF here.

ICW Assay Webinar

Amy Schutz-Geschwender, Ph.D. Principal Scientist, LI-COR Biosciences See Replay Here!

Application Summary

The In-Cell Western is an immunocytochemical assay performed in microplate format. Target-specific primary antibodies and infrared-labeled secondary antibodies are used to detect target proteins in fixed cells, and fluorescent signal from each well is quantified. Accuracy is enhanced and data are more meaningful because proteins are detected in their cellular context.

Application Overview

The unique advantages of infrared fluorescence allow In-Cell Westerns to provide extremely sensitive and quantitative analysis of cellular signaling pathways in cultured cells in a higher throughput manner. Use of infrared fluorescence reduces interference from cell, plate, and drug compound autofluorescence when compared to standard methods.

In-Cell Westerns simultaneously detect two targets at 700 and 800 nm using two spectrally distinct dyes. Separate lasers and fluorescence detectors are used for each dye and offer a wide linear detection range.

With two detection channels you can probe two separate targets or increase quantification accuracy by using the second channel for normalization against a second target or DNA stain. Quantification accuracy is maximized by normalization because adjustments can be made for differences in cell number from well to well. Two-color normalization also helps prevent false negatives and provides more accurate evaluation of cell treatment or drug candidate effects.

In-Cell Western Assay Workflow

Adherent Suspension

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