HTS / HCS Screening LiCor InCell Western Assay
Using DRAQ5™ and Sapphire700™ for Cell Number Normalization
For fuller information, please refer to the LiCor datasheet here.
DRAQ5™ and Sapphire700™ are included in the LiCor InCell Western assay kit and are designed to be used in combination, to provide accurate normalization over a broad range of cell densities.
DRAQ5™ is sold as part of this kit as a cell permeable DNA-interactive agent that can be used for stoichiometric staining of DNA in live or fixed cells. When serial dilutions of A431 human epithelial carcinoma cells are plated in 96-well plates, DRAQ5 demonstrates linearity of fluorescent signal for lower cell densities, up to ~50,000 cells/well, at the concentration used*.
Sapphire700™ is a non-specific cell stain that accumulates in both the nucleus and cytoplasm of fixed or dead cells, but not live cells. When used to stain serial dilutions of A431 cells in 96-well plates, Sapphire700 displays linearity of fluorescent signal for higher cell densities, from ~50,000 to ~250,000 cells/well.
Simultaneous staining of cells with both DRAQ5™ and Sapphire700™ expands the linear range, allowing more accurate normalization of cell number across both low and high cell densities.
In Cell Western assays commonly use primary and secondary antibodies for normalization in the 700 nm channel. For example, if phospho-ERK is the target of interest, an antibody against total ERK (or against a housekeeping protein) can be used to normalize for variations in cell number. Staining with DRAQ5™ and Sapphire700™ eliminates the need for this additional primary and secondary antibody, and yields the same quantitative measurement of ERK phosphorylation.
*Recent references have also described the use of DRAQ5 alone for this application, suggesting that higher concentrations of DRAQ5 are linear over a wider range than in the LiCOR kits:
- Hannoush RN (2008). Kinetics of Wnt-Driven b-Catenin Stabilization Revealed by Quantitative and Temporal Imaging. PLoS ONE 3(10): e3498. doi:10.1371/journal.pone.0003498
- Development of a mechanistic cell-based assay for the identification of JAK2 inhibitors. Richardson et al, Astex Therapeutics Ltd., Cambridge UK. Poster presentation at SBS-ELRIG's Drug Discovery 2008, September 23-24, 2008.
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