In-Cell Western™ is a simple and cost effective means for quantification of intracellular signaling in whole cells. This assay involves seeding cells in microtiter plates followed by fixation/permeabilization and subsequent labeling with activation state-specific or control antibodies, infrared-conjugated secondary antibodies, and/or far-red DNA dye. The well-level data acquired by the LI-COR® Odyssey® Infrared Imaging System permit accurate measurement of signal induction or inhibition, with the potential for multiple treatments, end points, and replicates on each plate.
Cell Signaling Technology (CST) offers over 1000 antibodies that may be used for In-Cell Western™ (ICW) assays. Because ICW assays are similar to classic immunocytochemistry, our antibodies that have been validated and approved for immunofluorescence (IF) are compatible with this assay. Simply follow our In-Cell Western™ Protocol to ensure correct labeling of your target proteins. If you have any questions, please contact our Technical Support team.
We offer secondary antibodies conjugated to DyLight® 680 or 800 near infrared fluorescent dyes. Due to their low background fluorescence, high sensitivity, photostability, and ease of quantification, DyLight® dyes are ideal for fluorescent western blotting and In-Cell Western™ (ICW) assays. Each CST DyLight® conjugated secondary antibody is tested in-house by fluorescent western and ICW analysis.
We also offer DRAQ5®, a near-infrared DNA dye. This dye can be used to normalize antibody signal to cell number, eliminating the potential for binding interference that may occur when multiplexing state-specific and total antibodies directed toward the same protein that have not been extensively tested for compatible use.