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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.
    Document Type:
    Reference
    Product Catalog Number:
    06-284
    Product Catalog Name:
    Anti-Nitrotyrosine Antibody

  • Identification and quantification of Gi-type GTP-binding proteins that copurify with a pituitary somatostatin receptor 8449959

    Somatostatin (SRIF) receptors of GH4C1 cells occupied with biotinyl-NH-[Leu8,D-Trp22,Tyr25] somatostatin28 (bio-S28) have been affinity purified over streptavidin affinity columns (Eppler, C. M., Zysk, J. R., Corbett, M., and Shieh, H.-M. (1992) J. Biol. Chem. 267, 15603-15612). This procedure results in the copurification of a single subtype of SRIF receptor (SSTR2) and associated guanine nucleotide-binding proteins (G proteins) that are coupled to these receptors. For accurate quantification it was necessary to: (i) use homogenous recombinant standards; (ii) accurately assess the purity of standards; (iii) determine recovery of G proteins during sample preparation and Western blotting; and (iv) account for cross-reactivity among antisera. Four pertussis toxin-sensitive G proteins were quantified with previously characterized polyclonal antisera. Gi alpha 1 also was measured with a novel, more sensitive monoclonal antibody (7H7). Go alpha and Gi alpha 2 but not Gi alpha 1 and Gi alpha 3 were detected in membrane extracts prepared from GH4C1 cells. In contrast, the G proteins copurified with SSTR2 receptors were predominantly Gi alpha 2 (50% of total G protein) and Gi alpha 3 (36% of total G protein), whereas Go alpha and Gi alpha 1 were negligible. G beta subunits also were detected. Silver staining confirmed the absence of a 39-kDa protein, corresponding to the M(r) of Go alpha associated with purified SRIF receptor-G protein complexes. These data suggest that SRIF receptors selectively couple to two G proteins, one of which is sparsely expressed in GH4C1 cells; the data conform to the notion that SRIF receptors discriminate between similar pertussis toxin-sensitive G proteins.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • 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.
    Document Type:
    Reference
    Product Catalog Number:
    2100
    Product Catalog Name:
    Protein-Concentrate Kit (Micro)

  • Considerations when measuring urinary albumin: precision, substances that may interfere, and conditions for sample storage. 1764788

    The measurement of small but abnormal amounts of albumin in urine is important in evaluating kidney disease in people with diabetes mellitus, hypertension, or possible adverse health effects from exposure to nephrotoxins. Routine laboratory methods for measuring albumin are not sensitive enough to measure the amounts that are significant in urine (less than 30 mg/L). In our laboratory we used three immunoassays for measuring urinary albumin: enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), and immunoturbidimetric assay (IT). We calculated the CVs of the three methods, investigated potential interfering substances at three times their normal concentrations, and stored urine under different conditions to find the best way to protect the sample until assay. The potential interferents we checked were transferrin, urea, beta 2-microglobulin, retinol-binding protein, creatinine, kappa and lambda light chains, IgG, hemoglobin, ketone, and glucose. The stability study involved two study temperatures (-20 and -70 degrees C) and four treatments (centrifuging or filtering, before or after storage). We found the following: the RIA had the lowest CV; the results from the interference study showed no interference from normal physiological concentrations of the substances investigated; storage at -70 degrees C regardless of the treatment should be adequate to prevent loss of albumin immunoreactivity.
    Document Type:
    Reference
    Product Catalog Number:
    20-176
    Product Catalog Name:
    100X GTPγS, 10mM
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