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Technical Support

Our scientists are at the bench daily to produce and validate our antibodies, so they have hands-on experience and knowledge of each antibody’s performance.

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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.

PathScan® Multi-Target HCA Kits

PathScan® Multi-Target HCA Kits allow the user to assay eight signaling molecules grouped by pathway or biological process.

7103 Well Schematic

Schematic representation of a potential 96-well plate layout for the PathScan® Multi-Target HCA Stress and Apoptosis Kit #7103. On this generic map, four treatments are performed in triplicate down the columns of the plate, while each of the eight antibodies is applied across individual rows of the 96-well plate. This layout is designed to allow the investigator to monitor the signaling of cellular stress and apoptotic pathways on one 96-well plate. The diagram above is one example; users may wish to reorganize plate map according to their needs.

7103 Graph

A549 cells were left untreated (blue) or treated with 25 μg/ml anisomycin (red) or 1 μM staurosporine (green). Mean fluorescence intensity was measured for antibodies in the PathScan® Multi-Target HCA Stress and Apoptosis Kit #7103. Data was generated on the Acumen® HCS platform.

ICW-Validated Antibodies

A wide array of Cell Signaling Technology (CST) antibodies have been validated for use in In-Cell Western™ (ICW) assays. Because ICW assays are similar to classic immunocytochemistry, antibodies that have been validated and approved for immunofluorescence cytometry (IF-IC) can be compatible with this assay. These antibodies can be identified using the applications field of the CST website and catalog. Whether the LI-COR® platform can successfully detect signal from the antibody depends on the protein expression level, which is usually cell line- and treatment-specific to a particular assay. If you have any questions, please contact our Technical Support team.

ICW Reagents

DyLight® Conjugated Secondary Antibodies

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.

DRAQ5®

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.