Figure 1. Methanol out performs Triton X-100 for detection of phospho-Stat3. A) Cells fixed with formaldehyde and permeabilized with Triton X-100. Positive and negative controls overlapped, indicating a poor assay for HTP screening (Z′ = -0.1). B) Cells were fixed and permeabilized with methanol. Wide seperation was observed between positive and negative controls, indicating a robust assay for screening (Z′ = 1.7).
Permeabilization is often required to provide access to the antigen, especially when aldehydes are used for fixation.
- When choosing a permeabilization reagent, subcellular localization should be considered.
- For detection of cell surface proteins, permeabilization may not be needed. See On-Cell Western Assays
Comparison of permeabilization methods for detection of phospho-Stat3 (Fig. 1)
Z′ factor is a statistic that measures the reproducibility of an assay for high-throughput screening. It is used to determine which data points are significantly different from the negative controls.
- Z′ is calculated by running a large number of positive and negative controls, and determining how much separation there is between positive and negative controls. If they overlap, or nearly overlap (Z′ < 0.5) due to large amounts of variation within the controls, the assay is useless for screening.
- An excellent assay exhibits good separation between positive and negative controls (Z′ > 0.5).1-3
- 1. Zhang, JH et al. J Biomol Screen 4: 67-73 (1999)
- 2. Hoffman, GR et al. Assay Drug Dev Tech 8(2): 186-99 (2010)
- 3. Boveia, V et al. LI-COR Application Note (2009)