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LI-COR REAGENTS
In-Cell Western™ Reagents
Reagents for LI-COR Imaging Systems
In-Cell Western™ Assay Kits
For Fast, Cost-Effective Cell-based Western Assays
The In-Cell Western™ Assay is an immunocytochemical assay that uses NIR fluorescence to detect and quantify proteins in fixed cells. Detecting proteins in their cellular context increases quantification precision. Proteins in fixed, cultured cells are detected directly in microplates, which yields higher throughput compared to Western blotting and eliminates typical Western blotting steps such as cell lysate preparation, electrophoresis, and membrane transfer. Using the In-Cell Western Assay kits, the cost per well for secondary screening is reduced to a fraction of the cost of typical screening methods.
The In-Cell Western™ Assay Kits provide antibodies, blocking buffer and stains for forty 96-well plates or ten 384-well plates. Sapphire700™ and DRAQ5™ protein stains are included to normalize for well-to-well differences in cell number. Using protein stains reduces the cost per assay compared to performing the assay using two secondary antibodies. Any potential interference caused by using two antibodies is also eliminated.
Sapphire700 Stain
Sapphire700 is a near-infrared fluorescent stain that is excluded from live cells with intact plasma membranes, but accumulates in cells that have lost membrane integrity, including cells that have been fixed in preparation for In Cell Western detection.Sapphire700 is used in combination with DRAQ5™ (Biostatus Limited) to normalize for cell number in In-Cell Western™ immunofluorescent assays.
Read about Sapphire700 in the literature in:
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Chernyavsky, AI et al.
Coupling of ionic events to protein kinase signaling cascades upon activation of alpha7 nicotinic receptor: cooperative regulation of alpha2-integrin expression and Rho kinase activity.
J Biol Chem. 284(33):22140-8 (2009)
Figure 1. Sapphire700 and DRAQ5 as normalizing agents for In-Cell Western assays. Dilutions of A431 cells were plated on clear, flat bottom 96-well plates, then fixed and permeabilized. Cells were stained with DRAQ5 alone, Sapphire700 alone, or both stains combined. A) Two-fold dilutions of cells, over a wide range of cell densities (0 - 200,000 cells/well). B) Closer examination of linearity of signal over the range of 25,000 - 250,000 cells/well, in dilution increments of 25,000 cells
Ordering Information
Please see our new Online Catalog for the latest ordering information.* Note: Additional DRAQ5 must be ordered from Biostatus Limited. Biostatus DRAQ5 is a 5 mM solution that requires 1:10,000 dilution rather than the 1:2000 dilution recommended for the 1 mM solution in LI-COR In-Cell Western Assay Kits I and II.
Shipping information:U.S.A.: Same-day shipping (Federal Express overnight) available on orders received before 12:00 noon CST.
Canada: Contact Mandel Scientific.
Germany & UK: Contact LI-COR GmbH.
International: Contact a LI-COR distributor in your area All shipments subject to availability.
