![]() ![]() The 520 nm channel with Revert™ 520 Total Protein Stain is shown on the left, and the 700 and 800 nm channels with pan-ERK1 and p-ERK1/2 bands are shown overlaid on the right. A) Western blot images for this assay are shown. EC 50 values were calculated that match expected results. Adapted with permission from Hoffman, GR et al. R 2 = 0.91, indicating exceptional plate-to-plate reproducibility. Plate-to-plate consistency is shown for a representative plate from the library. Known inhibitors of mTORC1 signaling found in the library are shown in green. Compounds with average Z-score < -2 are considered hits (red circles). A library of ~2500 known bioactive compounds was used. A pilot small molecule siRNA screen was performed for inhibitors of mTORC1 signaling. Replicability of In-Cell Western Assay for siRNA screening via a correlation plot. B) Signal intensities from In-Cell Western wells are plotted as a proportion of the mean values for each of two plates (plate 1 in blue, plate 2 in red). 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). A) GAPDH and PMLC20 were detected on duplicate Western blots (13 replicates per blot, not shown). Intra-assay variability of Western blots and In-Cell Western Assays. In-Cell Western Assays offered superior precision, reduced variability, and smaller CVs. In-Cell Western Assay and Western blot analysis yielded very similar results (Figure 1). 1 Primary cultures of uterine myocytes stimulated with oxytocin were used to assess specificity, sensitivity, and precision of the two methods for phospho-analysis. 2, 4, 5Ī 2010 study compared In-Cell Western Assays and Western blotting (WB) for measurement of phosphorylated myosin regulatory light chain (PMLC20). If they overlap, or nearly overlap (Z′ 0.5). 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. Z′-Factor can provide some indication of the replicability of an assay. Z′-Factor is a measure of statistical effect size and can be used to assess if a response to an assay requires further investigation. ![]() Produces excellent Z′-Factor with optimized conditions and experimental design.Very similar profiles of signal increase and decrease when compared to Westerns.Characterize a broad range of cell signaling parameters.Easily run many replicates to increase accuracy (Figure 2).Replicate measurements with very low coefficients of variation (CVs) (Figure 1).Significantly smaller standard deviations.In-Cell Western Assays exhibit the following characteristics: In-Cell Western Assays provide greater replicability and precision than Western blots. Target Analysis: Quantify the effect of a treatment or condition on a target.Z′-Factor Determination: Test the quality and robustness of an assay.Fixation and Permeabilization Evaluation: Determine the optimal fixation and permeabilization conditions for an experiment.Blocker Evaluation: Determine the best blocking buffer or blocking buffer and antibody combination for an experiment.Antibody Titration: Determine the antibody concentration that provides optimal signal and lowest background.Cell Stain Linearity: Ensure signal intensity for the normalization method and target are each detected within their linear range.For In-Cell Western Assays, create a custom template or use one of the six preset templates for: These straightforward and systematic workflows in Empiria Studio automate the critical steps of your analysis. Normalize to cell number, allowing for accurate quantification and comparison of protein expression between wellsĮmpiria Studio ® Software has step-by-step workflows and Plate Templates for various types of multiwell plate assays, including the In-Cell Western Assay.Detect proteins in situ in a relevant cellular context.Quickly, accurately measure relative protein levels in many samples.Quantify multiple targets using spectrally distinct fluorescent dye conjugates.Detect proteins in fixed and permeabilized cells using target-specific primary antibodies and IRDye ® Secondary Antibodies.In-Cell Western Assays are also called cytoblots, cell-based ELISA, In-Cell ELISA (ICE), and FACE (Fast Activated Cell-based ELISA). The In-Cell Western Assay is a quantitative immunofluorescence assay performed in multiwell plates (optimized for 96- or 384-well format) that combines the specificity of Western blotting with the replicability and throughput of ELISA. ![]()
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