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In-cell western assays also known as cell based assays allow researchers a simple and rapid assay method for the quantification biomarkers and signaling proteins in whole cells using antibodies. The Odyssey® infrared imaging system by LICOR® is well suited for ICW but other imaging systems may also be used. The method has many similarities to both Western blot and immunohistochemistry, and combines the specificity of a western blotting with the quantification of an ELISA. In-Cell Western assays are also described as cell-based ELISA, In-Cell ELISA (ICE), and FACE (Fast Activated Cell-based ELISA). ICW can be used for:
Figure 1. Example of In-Cell Western (ICW) or In-Cell ELISA (ICE) data. In this example, drug treated cells would be stained with 2 different antibodies, a control antibody (Dylight 700, RED), and a response biomarker protein (Dylight800, GREEN).
In the ICW method, cells are grown on a multi-well plate, typically 96- or 384-well microtiter plate. The cells may be untreated, or cells may be treated with drug, stimulant, or other agent. Cell culture studies, media formulation and cell banking are all part of cell culture services available from Rockland. Next the cells are prepared for staining by fixing and permeabilization. Antibodies are used to label control targets (i.e. GAPDH) as well as the desired protein of interest. Dye-conjugated secondary antibodies are used to detect the formed immune complex from each independent microtiter well. This allows for multiple data points, treatments, and replicates on each plate.
Many of Rockland Immunochemicals’ antibodies have been validated for use in In-Cell Western (ICW) assays. Since ICW assays are related to immunofluorescence most antibodies that have been validated and approved for these aforementioned immunoassays can be similarly used with ICW assays. Antibodies suitable for cell based immunoassays can be identified under the applications field of the Rockland website catalog. For assistance with your assay design or antibody selection, please contact firstname.lastname@example.org.Rockland offers secondary antibodies conjugated to IRDye®700, DyLight®680 and DyLight®800 near infrared fluorescent dyes. IRDyes® and DyLight® dyes are suitable for fluorescent western blotting and In-Cell Western (ICW) assays. Each IRDye® and DyLight® conjugated antibody is tested at Rockland by fluorescent western for specificity and sensitivity.
Near Infrared In Cell Western Antibodies
See all IR700 Antibodies
See all DyLight 680 Antibodies
See all Dylight 800 Antibodies
IR700 Antibodies611-130-002 Rabbit IgG (H&L) Secondary Antibody IRDye700DX® Conjugated610-430-002 Mouse IgG (H&L) Antibody IRDye700DX® Conjugated DyLight®680 Antibodies611-144-002 Rabbit IgG (H&L) Antibody DyLight™ 680 Conjugated610-144-002 Mouse IgG (H&L) Antibody Dylight™ 680 ConjugatedDyLight®800 Antibodies611-145-002 Rabbit IgG (H&L) Antibody DyLight™ 800 Conjugated610-145-002-0.5 Mouse IgG (H&L) Antibody Dylight™ 800 Conjugated
Rockland also offers control cell lysates and extracts and custom molecular biology services for the production of transformed cell lines and expression of recombinant proteins.
ReferencesJ Thromb Haemost. 2006 Mar;4(3):614-20. Egorina EM, Sovershaev MA, Østerud B.In-cell Western assay: a new approach to visualize tissue factor in human monocytes.IRDye™ is a trademark of LI-COR, Inc. Odyssey® is a registered trademark of LI-COR, Inc. DyLight™ is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries.
Rockland Immunochemicals Inc.Gilbertsville, PA 19525E-mail: email@example.comPhone: 800.656.7625