In-cell Westerns

What is an in-cell Western?
Cell-based ELISA, also called in-cell ELISA (ICE) or in-cell Western (ICW), is an immunocytochemistry method that combines the specificity of western blotting with the high-throughput of ELISA. The approach allows you to quantify proteins in cultured cells in situ, assessing the effects of drugs or other interventions on protein levels without further manipulation of the cells.

Traditional Western blotting can be time consuming. Protein samples must be extracted and electrophoresis and transfer steps must be conducted before antibody binding and detection can be carried out. When size information isn’t required, dot or slot blotting can save time by bypassing the electrophoresis step, and with a special apparatus many more samples can be analyzed on one blot than on a traditional Western, increasing throughput somewhat. However, before dot or slot blotting, protein extracts must be prepared from each cell type or condition being assessed.

ELISA (enzyme-linked immunoassay) provides a high-throughput approach to quantifying the presence of a protein or other antigen in a sample. ELISA relies on antibodies specific to the protein or antigen of interest coating the bottom of the wells of a 96- or 384-well plate to quickly capture the target when samples are added to the wells. However, an ELISA also requires extracting proteins from the cells of interest which increases the time to obtain results, and also introduces the potential to lose or alter the antigen of interest during the cell harvesting, lysing, and other extraction steps.

In-cell Westerns turn ELISA on its head, with cells (target) bound to the bottom of the wells of a 96- or 384-well plate rather than an antibody. The cells are fixed and permeabilized in the culture dish where they were grown, and then the target protein is detected much like in a traditional Western blot. The cells are blocked to prevent nonspecific binding, incubated with the primary antibody, washed and then incubated with a labeled, secondary antibody.

ELISAs cell-based-western

For in-cell Westerns, the secondary antibody is usually labeled with a near-infrared (NIR) fluorophore, which is less subject to interference from auto-fluorescence or from noise from the plastic of the tissue culture dish. A nuclear stain can be used to normalize the signal from the Western to the number of cells in the well. Cell-based ELISAs/in-cell Westerns therefore provide an excellent, high-throughput way to examine how growth conditions, drugs, or other interventions change protein levels in cultured cells.

Ready to start with in-cell Westerns? The Azure Sapphire Biomolecular Imager is an imager capable of in-cell westerns due to its sensitivity and speed. With four fluorescent channels including two NIR channels, the Sapphire facilitates multiplex detection like a pro.

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Document TypeDescription
Application NoteIn-cell Western BlottingDOWNLOAD

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