Luminescence assays

Fig. 8.2 Gel filtration on a column of Sephadex G-100 at pH 8 (both panels) of the crude extract of Gonyaulax polyedra cells prepared at pH 8 (upper panel) and prepared at pH 6 (lower panel). The activities of the 35 kDa and 130 kDa luciferases are measured by the addition of an excess of luciferin at pH 6.3 ( ) or at pH 8(A). The activity of the luciferin-bound LBP (luciferin-binding protein) in the upper panel is measured after the addition of an excess of 35 kDa luciferase at pH 6.3 ( ). In the lower panel, the LBP activity can be obtained by the addition of an excess of luciferin at pH 8, followed by the removal of unbound luciferin with a small column of Sephadex G-25 before the luminescence assay of bound luciferin at pH 6.3 (see the Section 8.2.8). The Overlap in the upper panel is the light emission resulting from the mixing of an aliquot of the fractions with pH 6.3 buffer. From Fogel and Hastings, 1971, with permission from Elsevier.

Argus Laboratories Ltd. Chemiluminescent and bioluminescent products Berthold. Luminescence assays http / / www.berthold-online.com/ http //www.net-escape.co.uk/business/argus/... 

Didenko, V.V. and Hornsby, P.J. (1996) A quantitative luminescence assay for nonradioactive nucleic acid probes. Journal of the Histochemistry Society, 44 (6), 657-660. 

Handl, H. L., Gillies, R. J. Lanthanide-based luminescent assays for ligand-receptor interactions. Life Sci. 2005, 77, 361-671. 

Cell proliferation GI s of the test agent should be determined in preliminary studies using assays compatible with 3D cultures (e.g., CellTiter Glo luminescent assay or WST-1 assay) so that appropriate concentrations can be selected. 

For this functional assay we use the Beta-Glo assay (Promega), a luminescent assay able to quantify Beta-Gal expression. Other methods allowing a sensitive measurement of the reporter could be adopted (see Note 11). 

Kay It is like flies. Their lateral neuron rhythms will damp 99% of the signal in the peripheral tissues. If you take those flies from the topcount luminescence assay and put them in locomotor and DD, you will see that their locomotor rhythms are still fine. It is not just a culture effect that you see damping of these rhythms. 

Bronstein, I. and Kricka, L. J. (1989) Clinical applications of luminescent assays for enzymes and enzyme labels. J Clin Lab. Anal 3,316-322. 

The technique described above is not useful for removing colored peroxidase reaction products (e.g., diaminobenzidine oxidation products) from blots Thus, we avoid detection methods based on these reactions. On the other hand, peroxidase-based luminescent assays (20) do not deposit a chemical reaction product on the blot and are compatible with this erasure method (21). An example of the use of this erasure method (Section 3 3, steps 1-5) after chemiluminescent detection is shown in Fig 2. 

In addition to the choice of QSAR descriptors, many statistical learning methods are available to relate the descriptors to the predicted value. There are two main categories of prediction to answer two different questions regression models (for predicting the activity of a peptide as a continuous variable such as MIC or a surrogate such as in the luminescence assay) or classification where the model is trained to classify as simply active or inactive. Historically, linear... 

Pal R, Parker D, Costello LC (2009) A europium luminescence assay of lactate and citrate in biological fluids. Org Biomol Chem 7 1525-1528... 

Effect of glutamine-leucine and phenylalanine-tyrosine substitutions on antibody binding. Peptides spotted onto cellulose membranes. Antibody binding measured in a luminescence assay [127]. At least 39 sera of CD patients positive for endomysium antibodies tested. 

Photomultiplier tube (PMT) detection. The photon counts from each well are measured one at a time by the PMT and the excitation is achieved through an excitation beam or laser. Radioactive assays and luminescent assays do not require excitation beams. Because radioactive assays require long counting times using PMT-based systems, these counters often accomplish throughput by running many PMTs in parallel. 

The selection of the type of assay plate is also dependent on the type of signal(s) to be detected and whether a clear bottom is required to observe cells under a microscope. Opaque black plates are often chosen for fluorescent assays and white plates are best for luminescent assays because of... 

Poole, R.A., Kielar, R, Richardson, S.L., et al. (2006) A ratiometric and non-enzymatic luminescence assay for uric acid differential quenching of lanthanide excited states by anti-oxidants. Chemical Communications, 4084-4086. 

FIGURE 14. Principle of the time-resolved (gated) luminescence assay. Reproduced with permission from Reference 2, Copyright 2005 the Royal Society of Chemistry... 

Thorpe, G.H.G. Moseley, S.B. Kricka, L.J. Stott, R.A. Whitehead, T.P. Enhanced luminescence determination of horseradish peroxidase conjugates apphcation of ben-zothiazole derivatives as enhancers in luminescence assays on microtitre plates. Anal. Chim. Acta. 1985, 170, 101. 

Several additional projects evolved from the initial TNAP project. Thanks to the broad dynamic range of the luminescent assay, several TNAP activator scaffolds were observed during HTS, resulting in an independent project with a potential therapeutic indication for osteomalacia and osteoporosis. Other human APs, intestinal (lAP) and placental (PLAP) isozymes, were initially utilized to establish counter screen assays based on CDP-star. One of the TNAP scaffolds demonstrated inhibition of lAP, and was further optimized to produce a selective chemical probe for human lAP. Later, the assays for lAP and PLAP were utilized for full-scale screening of these isozymes, eventually leading to identification of selective inhibitors for both isozymes. Interestingly, human lAP... 

These compounds had simple but highly brominated structures. Authentic TBHQ was subjected to the luminescence assay and showed an almost equivalent intensity as the isolated Fr.l. This result confirmed that TBHQ emitted the light. 

The development of automated luminometers is focused primarily on devices that achieve high sample throughput rates, typically through the use of 96 and 384 well microtitre plates. Whilst suited for many applications, these systems require a skilled operator and often do not provide rapid results when the time taken to prepare the microtitre plate is included in the assay time. An instrument that can perform on demand, automated, near real time analysis using a variety of luminescent assay protocols has been developed. The instrument has been designed to automate various luminescent assays including adenylate kinase (AK) assays and those that use magnetic separation steps in conjunction with bioluminescence. ... 

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