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  • Rapid and sensitive determination of protein-nitrotyrosine by ELISA: Application to human plasma. 22276750

    3-Nitrotyrosine (3NT) is known as an important indicator of nitrosative stress and has been linked to various diseases. Our aim was to develop an indirect ELISA (enzyme-linked immunosorbent assay) method suitable for the detection of protein-bound 3NT in clinical plasma and serum samples. Nitrated protein standards and reduced protein standards were prepared. Limit of detection was determined for standards; recovery and reproducibility were determined for human plasma samples. The limit of detection for this method is 1.82±0.56 pmol/mg protein. Mean recovery of standards was 95%. 3NT concentration in plasma samples of obese and normal weight subjects was determined to be between 2 pmol/mg and 19 pmol/mg. No time-consuming sample preparation or expensive laboratory equipment is required, and applied antibodies are commercially available. Sensitivity, rapid analysis time, possibilities of high throughput applications and small sample volumes make this ELISA attractive for use in clinical laboratories.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    06-284
    Produktbezeichnung:
    Anti-Nitrotyrosine Antibody

  • Large-Scale Filter-Aided Sample Preparation Method for the Analysis of the Ubiquitinome. 28260372

    Protein ubiquitination regulates key cellular functions, including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-ε-GG enrichment. Filter-aided sample preparation (FASP) surpasses in-solution protein digestion in cleavage efficiency, but its performance has thus far been shown for digestion of sample amounts on the order of micrograms. Because cleavage efficiency is pivotal in the generation of the K-ε-GG epitope recognized during IAP, here we developed a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASP-based tryptic digestion is efficient, robust, reproducible, and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show a ∼3-fold reduction in the proportion of miscleaved peptides with the method presented here. Beyond ubiquitinome analysis, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a variety of applications that demand a large(r) amount of starting material.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    UFC901024
    Produktbezeichnung:
    Amicon Ultra-15, PLGC Ultracel-PL Membran, 10 kDa

  • Paraoxonase-3, a Putative Circulating Antioxidant, Is Systemically Up-Regulated in Late Gestation in the Fetal Rat, Sheep, and Human. 20463093

    Context: Surfactant is a successful therapeutic based on supplementing preterm infants with a substance that would normally have been up-regulated in late gestation. Although prematurity is associated with oxidative stress, no effective antioxidant therapy has yet been identified. Objective: Our objective was to identify endogenous antioxidants involved in fetal preparation for birth. Design: We performed transcript profiling of fetal rat lung and intestine at 16 d gestational age (dGA) and 20 dGA with out-of-sample validation. Gene expression was then measured in fetal sheep tissues, comparing 1) advancing GA, 2) exogenous maternal dexamethasone (compared with saline, at 130 dGA), and 3) fetal adrenalectomy at 115-118 d on levels at term. Protein levels were compared in human umbilical cord serum using Western blot. Results: Four transcripts were up-regulated more than 20-fold on the array in both rat lung and intestine. One of these, paraoxonase-3 (Pon3), had been identified as a putative circulating antioxidant. Up-regulation of Pon3 mRNA in rat lung, intestine, and liver was confirmed in siblings (all P < 0.001). Pon3 mRNA levels in fetal sheep lung and intestine increased 5.1- and 5.3-fold, respectively (both P < 0.001) between 100 and 145 dGA and were strongly correlated with plasma cortisol (both P < 0.001). Fetal sheep pulmonary Pon3 transcript level was increased 55% (P = 0.01) by dexamethasone and reduced 74% (P < 0.001) by adrenalectomy. Term human infants had more than 6-fold higher umbilical cord serum levels of Pon3 than preterm (24-28 wk GA) infants (P < 0.001). Conclusions: Pon3, a putative circulating antioxidant, was systemically up-regulated in late-gestation rat, sheep, and human fetuses and is a candidate therapeutic in preterm human infants.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    2100
    Produktbezeichnung:
    Protein-Concentrate Kit (Micro)

  • Homocysteine and other thiols determined in plasma by HPLC and thiol-specific postcolumn derivatization. 8353942

    We describe a versatile high-performance liquid-chromatographic method for determining homocysteine and other plasma sulfhydryls. Using three different procedures for preparation of plasma, we determined total, free (non-protein-bound), and reduced forms of homocysteine, cysteine, glutathione, cysteinylglycine, and gamma-glutamylcysteine in human plasma. Sample preparation involves disulfide reduction with dithiothreitol and protein precipitation with sulfosalicylic acid. The assay utilizes isocratic reversed-phase ion-pair liquid chromatography at pH 2.4, postcolumn derivatization with 4,4'-dithiodipyridine, and colorimetric detection at 324 nm. The intra-assay precision (CV) of the method for total homocysteine is 1.5%; the interassay precision over 2.5 months is 2.5%. The detection limit for homocysteine is < 50 nmol/L plasma.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    20-176
    Produktbezeichnung:
    100X GTPγS, 10mM
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