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High sensitive detection of follistatin by Imperacer®: an initial study on the way to analytical gene-doping tests
For the understanding of interactions in the muscle-growth directing myostatin-pathway and therefore also for the establishment of high sensitive detection strategies for gene-doping, detailed studies of protein concentration levels in biological matrices and appropriate highly sensitive analytic techniques for quantitative monitoring of these biomarkers are required. In this work, the development of a high sensitive assay for follistatin based on the Imperacer® technology is described. The quantification limit in human serum was found at 60 pg/ml in only 6 µl sample volume. With this initial assay we demonstrate the first application in a set of analytical tests for a high sensitive myostatin pathway related profiling....
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Molecular Probes® NanoOrange® Assay Performed on BMG LABTECH FLUOstar OPTIMA Microplate Reader
The field of proteomics has expanded dramatically in recent years with research on a whole host of organisms. Techniques such as two dimensional difference gel electrophoresis (2D DIGE) require the accurate quantitation of protein, as up to three labelled samples can be loaded on a single gel for comparison. Assay methods for determining protein quantitation include absorbance at 280 nm(1), the Bradford assay(2), Lowry assay(3), BCA method(4) and more sensitive assays such as Fluoroprofile® (Sigma-Aldrich) and NanoOrange® (Molecular Probes®)5()....
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Rapid High-Throughput Methylation Analysis Using the LightCycler® 480 System
The LightCycler® 480 Instrument is a versatile platform for molecular studies. We assessed the performance of the instrument combined with the LightCycler® 480 High Resolution Melting Master mix for DNA methylation analysis using the methylation-sensitive high resolution melting (MS-HRM) methodology. This proved to be a robust platform for performing MS-HRM experiments, allowing rapid and high-throughput studies of DNA methylation....
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Quantitative Chromatin Immunoprecipitation Using the LightCycler® 480 System
Antigen processing and presentation play a critical role in immune surveillance. Peptides derived from self, viral, or tumor-associated antigens are generated in the cytoplasm by the proteasome. Transporter associated with antigen processing (TAP), a member of the ATP-binding cassette (ABC) transporter family, functions by transporting these peptides from the cytoplasm to the lumen of the endoplasmic reticulum (ER), where each peptide forms a ternary complex with beta-2 microglobulin (ß2m) and major histocompatibility complex (MHC) class I heavy chain. These complexes are then transported to the cell surface and recognized by cytotoxic T lymphocytes (CTLs), which eventually kill cells that present non-self antigens....
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Methylation of the Arginine 2 in Histone H3 Controls Deposition of Lysine 4 Tri-Methylation
In a recent study, Kirmizis et al. demonstrated that H3R2 is methylated not only in mammals but also in Saccharomyces cerevisiae. By using a specific H3R2me2a antibody in a ChIP-on-Chip analysis the authors determined the distribution of the methylation throughout the yeast genome, finding an enrichment of H3R2me2a at all heterochromatic genes, at inactive euchromatic genes, and at the 3“-end of moderately transcribed genes. The presence of H3R2 methylation inversely correlates with the presence of H3K4 tri-methylation (H3K4me3) in all cases, reflecting the fact that H3R2 methylation disrupts the ability of the Set1-complex to methylate H3K4 by preventing the Set1-methyltransferase subunit Spp1 from binding to to histone H3. These results show that H3R2 methylation controls the distribution of H3K4me3 and provide the first mechanistic insight into the function of arginine methylation on chromatin....
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