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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
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    C5737
    Produktbezeichnung:
    ZipTip® Pipette Tips

  • 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

  • Cross-sample validation provides enhanced proteome coverage in rat vocal fold mucosa. 21423617

    The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high M(r) ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high M(r) glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    AB1954
    Produktbezeichnung:
    Anti-Fibronectin Antibody

  • Spinning-disk confocal microscopy of yeast. 20946826

    Spinning-disk confocal microscopy is an imaging technique that combines the out-of-focus light rejection of confocal microscopy with the high sensitivity of wide-field microscopy. Because of its unique features, it is well suited to high-resolution imaging of yeast and other small cells. Elimination of out-of-focus light significantly improves the image contrast and signal-to-noise ratio, making it easier to resolve and quantitate small, dim structures in the cell. These features make spinning-disk confocal microscopy an excellent technique for studying protein localization and dynamics in yeast. In this review, I describe the rationale behind using spinning-disk confocal imaging for yeast, hardware considerations when assembling a spinning-disk confocal scope, and methods for strain preparation and imaging. In particular, I discuss choices of objective lens and camera, choice of fluorescent proteins for tagging yeast genes, and methods for sample preparation.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • 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.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere
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