Notes: The authors of this study explore the hypothesis that mitochondria pass glycolytic enzymes to the nucleus during early development to assist with producing substrates needed for epigenetic changes needed for activating the zygotic genome. Embryo extracts were analyzed with the ENLITEN® ATP Assay and the ADP-Glo™ Assay for ATP:ADP ratio; glutathione levels monitored with GSH-Glo™ Assay.  Extracts were also made as directed in the NAD/NADH-Glo™ Assay manual to determine NAD and NADH levels. (4831)

Notes: This article describes how a miniaturized high-throughput biochemical assay for Yes1 kinase was developed and used for screening inhibitors in small molecule libraries. Kinase activity of Yes1 was assessed by dispensing 2µl of Yes1 enzyme in a 1,536-well white multiwell plate and mixing with compounds and substrate for a total kinase reaction volume of 25µl. The amount of ADP was quantitated using the ADP-Glo™ Kinase Assay and normalized to vehicle and minus-enzyme controls. Some of the inhibitors identified in the in vitro assay were tested in a cell-based assay. Two rhabdomyosarcoma cell lines, RD and RH30, were seeded at 4,000 cells/well in a 96-well plate with 100µl of culture medium. After an overnight incubation, 100µl of medium with 0, 0.1, 1, 5, 10, 15 or 20μM of inhibitor (final concentration) was added. After 48 hours, the number of cells was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. (4354)

Notes: The authors of this paper investigated mutations affecting the hinge loop of protein kinases that appear to confer resistance to both Type I and Type II inhibitors. They introduced individual amino acid substitutions into the hinge region of six distantly related protein kinases and determined the inhibitor sensitivity of these kinases. The ADP-Glo™ Kinase Assay was used to asses the activity of the Haspin and c-Src kinases and the engineered mutants in this study. (4144)

Notes: The authors of this study evaluated the ADP-Glo™ Assay technology for use in high-throughput screening applications for inhibitors of all four known mammalian PI 4-kinases. They found that K_m values, IC₅₀ values of known inhibitors, and dose-response curves were comparable to values reported in the literature or those obtained using the standard isotopic assay. Z´-factor values for the assay in a low-volume, 384-well format were 0.72 and 0.74, indicating that the assay would be suitable for screening activities in 384- or 1536-well formats. (4129)

Notes: This paper describes the use of the ADP-Glo™ Assay technology to screen for small-molecule inhibitors of PI 4-kinases (phatidylinositol 4-kinases). The authors characterized E. coli-expressed proteins using the ADP-Glo™ Kinase Assay and saw strong signal-to-background ratios, no signal from their negative controls or the D1899A protein, and no signal in the absence of micelles. Next they compared data from the ADP-Glo™ Assay with results obtained from the standard isotopic method or results available in the literature. They were able to obtain K_m values for PI4KA and PI4KB that corresponded to published values. Finally, the authors evaluated the potential of the ADP-Glo™ Assay as a high-throughput screening tool. To assess the assay, they used Z´ factor as their statistical measure, which is a description of the dynamic range and the variability of the assay. In this test, the Z´ factor values were 0.72 and 0.74. From these results, the authors conclude that the assay would work well for screening in a 384-well system and could likely be optimized for screening in a 1536-well format. (4135)

Notes: The authors of this paper demonstrate the utility of the HaloTag^® protein purification system for purifying functional proteins from mammalian cells. To this end five kinases were cloned into HaloTag^® vectors, expressed in and purified from HEK293T cells. To demonstrate functionality of the purified recombinant kinases, activity was measured using the appropriate ADP-Glo™ Assay Kinase Enzyme Systems. (4145)