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A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors.

ABSTRACT: Non-radioactive assays based on incorporation of puromycin into newly synthesized proteins and subsequent detection using anti-puromycin antibodies have been previously reported and well-validated. To develop a moderate- to high-throughput assay, an adaptation is here described wherein cells are puromycin-labeled followed by simultaneously probing puromycin-labeled proteins and a reference protein in situ. Detection using a pair of near IR-labeled secondary antibodies (InCell western, ICW format) allows quantitative analysis of protein synthesis in 384-well plates. After optimization, ICW results were compared to western blot analysis using cycloheximide as a model protein synthesis inhibitor and showed comparable results. The method was then applied to several protein synthesis inhibitors and revealed good correlation between potency as protein synthesis inhibitors to their ability to sensitize TRAIL-resistant renal carcinoma cells to TRAIL-induced apoptosis.

SUBMITTER: Henrich CJ

PROVIDER: S-EPMC5074477 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors.

Henrich Curtis J CJ

PloS one 20161021 10

Non-radioactive assays based on incorporation of puromycin into newly synthesized proteins and subsequent detection using anti-puromycin antibodies have been previously reported and well-validated. To develop a moderate- to high-throughput assay, an adaptation is here described wherein cells are puromycin-labeled followed by simultaneously probing puromycin-labeled proteins and a reference protein in situ. Detection using a pair of near IR-labeled secondary antibodies (InCell western, ICW format ...[more]

PMID: 27768779

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Project description:AimsThe well-documented relationship between sperm oxidation and male infertility strongly encourages the development of assays for reactive oxygen species detection in semen samples. The present study aims to apply the microplate-based 2',7'-dichlorofluorescein diacetate assay to the evaluation of oxidative stress in unprocessed whole semen, thus avoiding sample centrifugations and other manipulations that may cause significant reactive oxygen species increments.Main methodsThe fluorescence assay consisted in the quantification of both intracellular and extracellular reactive oxygen species levels in unwashed semen specimens by using the probe 2',7'-dichlorofluorescein diacetate into a 96-well plate. The method was useful for the preliminary assessment of the oxidation levels of whole semen samples from men undergoing standard sperm analysis as well as to evaluate the effect of some pro-glutathione molecules on semen oxidative status.Key findingsThe 2',7'-dichlorofluorescein diacetate assay was successfully adapted to the evaluation of oxidative stress in whole semen, effectively revealing the perturbation of the redox homeostasis of the sample. Accordingly, specimens with abnormal sperm parameters (n = 10) presented oxidation indexes significantly higher than those with normospermia (n = 10) [7729 (range 3407-12769) vs. 1356 (range 470-2711), p < 0.001]; in addition, semen oxidation indexes negatively correlated to sperm motility and morphology. Noteworthy, whole semen exposure to pro-glutathione compounds led to reduced semen oxidation levels and sperm protection against oxidative damage.SignificanceBased on our pilot experimental data, the microplate-based 2',7'-dichlorofluorescein diacetate assay appears to be a convenient method for the detection of reactive oxygen species levels in whole semen samples, avoiding artifacts due to semen centrifugation steps. At the same time, the test could be a helpful tool for the basic and quick screening of antioxidant molecules able to preserve semen quality.

Dataset Information

A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors.

Publications

A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors.

Similar Datasets

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