Figure 1. Intra-plate variability of ICW is very low. HeLa cells were treated with 10 μm chloramphenicol (CAM) or vehicle (DMSO) for 7 days. Levels of OXPHOS Complex IV (COX-1, MS404; Mitosciences) were analyzed by In-Cell Western. Statistics are shown for background (no primary antibody; n=16), DMSO, and CAM treatments (n=40 for each; CVs = 6% and 4%, respectively).
Figure 3. Reproducibility of ICW for siRNA screening. A pilot small molecule siRNA screen was performed for inhibitors of mTORC1 signaling. A library of ~2500 known bioactive compounds was used. Compounds with average Z-score < -2 are considered hits (red circles). Known inhibitors of mTORC1 signaling found in the library are shown in green. Plate-to-plate reproducibility is shown for a representative plate from the library. Adapted with permission from Hoffman, GR et al.2
In-Cell Western assays provide greater reproducibility and precision than Western blots:
Figure 2. Intra-assay variability of Western blots and ICW assays. A) GAPDH and PMLC20 were detected on duplicate Western blots (13 replicates per blot; not shown). Band intensities are expressed as a proportion of the mean of the thirteen samples on each blot (blot 1 in blue, blot 2 in red). CV was 0.27. B) Signal intensities from ICW wells are plotted as a proportion of the mean values for each of two plates (plate 1 in blue, plate 2 in red). CV ranged from 0.08 to 0.16. Aguilar, HN et al. PLoS ONE 5(4): e9965 (2010). doi:10.1371/journal.pone.00099
A 2010 study compared ICW Assays and Western blotting (WB) for measurement of phosphorylated myosin regulatory light chain (PMLC20).1 Primary cultures of uterine myocytes stimulated with oxytocin were used to assess specificity, sensitivity, and precision of the two methods for phospho-analysis.
In-Cell Western assay and Western blot analysis yielded very similar results (Fig. 2). ICW assays offered superior precision, reduced variability, and smaller CVs.