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.
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.
PhosphoPlus® In-Cell Duets
PhosphoPlus® In-Cell Duet Kits allow levels of phosphorylated protein to be independently measured and normalized.
Analysis of MDA-MB-468 cells exposed to varying concentrations of LY294002 (PI3 Kinase Inhibitor) #9901 for 2 hours, followed by hEGF #8916 stimulation for 20 minutes. The phosphorylation status of Akt, as well as the total protein expression level, was measured simultaneously using the PhosphoPlus® Akt (Ser473) In-Cell Duet (ICW Compatible) #7255. With increasing concentrations of LY294002, a significant decrease (~3-fold) in phospho-Akt signal as compared to the hEGF-stimulated control was observed, while total Akt protein levels remained the same and were used to normalize the data. When using phospho-Akt as a measurement, the IC50 of this compound was 2.7 μM. Data and images were generated on the LI-COR® Biosciences Odyssey® Infrared Imaging System.
PathScan® Multi-Target HCA Kits
PathScan® Multi-Target HCA Kits allow the user to assay eight signaling molecules grouped by pathway or biological process.
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.
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.
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. For assistance with your assay design or antibody selection, please contact ICW@cellsignal.com.
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.
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.