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  • GEL-FREE SAMPLE PREPARATION FOR THE NANOSCALE LC-MS/MS ANALYSIS AND IDENTIFICATION OF LOW-NANOGRAM PROTEIN SAMPLES 17763504

    Protein identification at the low nanogram level could in principle be obtained by most nanoscale LC-MS/MS systems. Nevertheless, the complex sample preparation procedures generally required in biological applications, and the consequent high risk of sample losses, very often hamper practical achievement of such low levels. In fact, the minimal amount of protein required for the identification from a gel band or spot, in general, largely exceeds the theoretical limit of identification reachable by nanoscale LC-MS/MS systems. A method for the identification of low levels of purified proteins, allowing limits of identification down to 1 ng when using standard bore, 75 microm id nanoscale LC-MS/MS systems is here reported. The method comprises an offline two-step sample cleanup, subsequent to protein digestion, which is designed to minimize sample losses, allows high flexibility in the choice of digestion conditions and delivers a highly purified peptide mixture even from "real world" digestion conditions, thus allowing the subsequent nanoscale LC-MS/MS analysis to be performed in automated, unattended operation for long series. The method can be applied to the characterization of low levels of affinity purified protei
    Document Type:
    Reference
    Product Catalog Number:
    C5737
    Product Catalog Name:
    ZipTip® Pipette Tips

  • Measurement of human surfactant protein-B turnover in vivo from tracheal aspirates using targeted proteomics. 20178338

    We describe a method to measure protein synthesis and catabolism in humans without prior purification and use the method to measure the turnover of surfactant protein-B (SP-B). SP-B, a lung-specific, hydrophobic protein essential for fetal-neonatal respiratory transition, is present in only picomolar quantities in tracheal aspirate samples and difficult to isolate for dynamic turnover studies using traditional in vivo tracer techniques. Using infusion of [5,5,5-(2)H(3)] leucine and a targeted proteomics method, we measured both the quantity and kinetics of SP-B tryptic peptides in tracheal aspirate samples of symptomatic newborn infants. The fractional synthetic rate (FSR) of SP-B measured using the most abundant proteolytic fragment, a 10 amino acid peptide from the carboxy-terminus of proSP-B (SPTGEWLPR), from the circulating leucine pool was 0.035 +/- 0.005 h(-1), and the fractional catabolic rate was 0.044 +/- 0.003 h(-1). This technique permits high-throughput and sensitive measurement of turnover of low abundance proteins with minimal sample preparation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • 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)

  • Chromatin immunoprecipitation (ChIP): revisiting the efficacy of sample preparation, sonication, quantification of sheared DNA, and analysis via PCR. 22046253

    The "quantitative" ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA) method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
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