Cytoblot assays

Acytoblotassay is a miniaturized cell-based assay format that interrogates the cellular pathways in mammalian systems using small molecules. This wholecell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Cytoblotting can be used to monitor bios5mthetic processes such as DNA synthesis, and posttranslational processes such as acetylation and phosphorylation. 

In the cytoblot assay, double immunochemical detection was applied the primary antibody detects, for example, phosphorylated proteins while a second antibody linked to horseradish peroxidase provides a chemiluminescence readout (Stockwell, Haggarty, and Schreiber, 1999). The applicability of the cytoblot assay was demonstrated by natural product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the antiproliferative effect of rapamycin. In another application, a similar setup was used to identify the phosphorylation pattern of multiple signaling proteins in response to known kinase inhibitors (Chen et al., 2005). 

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Mayer et al. (19) reported the use of FCG to identify compounds that affect the cellular mitotic process through a screening cascade reported in Fig. 9.2. A commercially available, diverse collection of 16,320 compounds was screened on a whole cell primary cytoblot assay (20) measuring the phosphorylation level of a nucleolar protein called nucleolin (step a). This protein is phosphorylated when cells enter mitosis, and inhibitors of the mitotic process are expected to increase the level of phosphonucleolin. 139 positive compounds were able both to penetrate the cell and to increase the level of phosphorylated nucleolin. 

Tubacin TDAC specific r x Histacin HDAC specific acetyl-tubulin cytoblot assay acetyl-histone cytoblot assay ... 

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