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Normalization to Cell Number:
Cell Labeling with Reactive Dye

fig1ReactiveDye

Figure 1. Use of cell labeling for In-Cell Western normalization. A) HeLa cells were treated with increasing amounts of rapamycin in a 384-well format. Fixed cells were stained with phospho-rpS6 antibody and NHS ester reactive dye (for cell number). Dose dependent inhibition of phospho-rpS6-staining yielded an IC50 of 224 pM (n=4). B) Raw microplate image. Data courtesy of GR Hoffman.1

  • Covalently label cellular lysine residues on cellular proteins with
    IRDye® 800CW (Fig. 1 and 2) or IRDye 700DX reactive dyes1
  • Very inexpensive method with extreme sensitivity and wide linear range1 (~200 to 200,000 cells/well) (Fig. 2)
  • Results are unaffected by changes in nuclear DNA
fig2ReactiveDye

Figure 2. Linearity of cell labeling with IRDye 800CW reactive dye. Two-fold serial dilutions of HeLa or A431 cells were fixed and permeabilized. Cells were labeled with IRDye 800CW NHS ester at a 1:50,000 dilution. The method was linear from ~200-200,000 cells/well.


  • 1. Hoffman, GR et al. Assay Drug Dev Tech 8(2):186-99 (2010)
  • Labeling of Fixed Cells with IRDye® NHS Ester Reactive Dyes for In-Cell Western™ Assay Normalization, Technical Note, LI-COR
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