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  • Profiling of the Chromatin-Associated Proteome Identifies HP1BP3 as a Novel Regulator of Cell Cycle Progression. 24830416

    The chromatin-associated proteome (chromatome) regulates cellular gene expression by restricting access of transcriptional machinery to template DNA, and dynamic re-modeling of chromatin structure is required to regulate critical cell functions including growth and replication, DNA repair and recombination, and oncogenic transformation in progression to cancer. Central to the control of these processes is efficient regulation of the host cell cycle, which is maintained by rapid changes in chromatin conformation during normal cycle progression. A global overview of chromatin protein organization is therefore essential to fully understand cell cycle regulation, but the influence of the chromatome and chromatin binding topology on host cell cycle progression remains poorly defined. Here we used partial MNase digestion together with iTRAQ-based high-throughput quantitative proteomics to quantify chromatin-associated proteins during interphase progression. We identified a total of 481 proteins with high confidence that were involved in chromatin-dependent events including transcriptional regulation, chromatin re-organization, and DNA replication and repair, while the quantitative data revealed the temporal interactions of these proteins with chromatin during interphase progression. When combined with biochemical and functional assays, these data revealed a strikingly dynamic association of protein HP1BP3 with the chromatin complex during different stages of interphase, and uncovered a novel regulatory role for this molecule in transcriptional regulation. We report that HP1BP3 protein maintains heterochromatin integrity during G1-S progression and regulates the duration of G1 phase to critically influence cell proliferative capacity.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1501
    Nombre del producto:
    Anti-Actin Antibody, clone C4
  • TDRD5 is required for retrotransposon silencing, chromatoid body assembly, and spermiogenesis in mice. 21383078

    The Tudor domain-containing proteins (TDRDs) are an evolutionarily conserved family of proteins involved in germ cell development. We show here that in mice, TDRD5 is a novel component of the intermitochondrial cements (IMCs) and the chromatoid bodies (CBs), which are cytoplasmic ribonucleoprotein granules involved in RNA processing for spermatogenesis. Tdrd5-deficient males are sterile because of spermiogenic arrest at the round spermatid stage, with occasional failure in meiotic prophase. Without TDRD5, IMCs and CBs are disorganized, with mislocalization of their key components, including TDRD1/6/7/9 and MIWI/MILI/MIWI2. In addition, Tdrd5-deficient germ cells fail to repress LINE-1 retrotransposons with DNA-demethylated promoters. Cyclic adenosine monophosphate response element modulator (CREM) and TRF2, key transcription factors for spermiogenesis, are expressed in Tdrd5-deficient round spermatids, but their targets, including Prm1/Prm2/Tnp1, are severely down-regulated, which indicates the importance of IMC/CB-mediated regulation for postmeiotic gene expression. Strikingly, Tdrd5-deficient round spermatids injected into oocytes contribute to fertile offspring, demonstrating that acquisition of a functional haploid genome may be uncoupled from TDRD5 function.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-636
    Nombre del producto:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301
  • Circadian Proteins CLOCK and BMAL1 in the Chromatoid Body, a RNA Processing Granule of Male Germ Cells. 22900038

    Spermatogenesis is a complex differentiation process that involves genetic and epigenetic regulation, sophisticated hormonal control, and extensive structural changes in male germ cells. RNA nuclear and cytoplasmic bodies appear to be critical for the progress of spermatogenesis. The chromatoid body (CB) is a cytoplasmic organelle playing an important role in RNA post-transcriptional and translation regulation during the late steps of germ cell differentiation. The CB is also important for fertility determination since mutations of genes encoding its components cause infertility by spermatogenesis arrest. Targeted ablation of the Bmal1 and Clock genes, which encode central regulators of the circadian clock also result in fertility defects caused by problems other than spermatogenesis alterations. We show that the circadian proteins CLOCK and BMAL1 are localized in the CB in a stage-specific manner of germ cells. Both BMAL1 and CLOCK proteins physically interact with the ATP-dependent DEAD-box RNA helicase MVH (mouse VASA homolog), a hallmark component of the CB. BMAL1 is differentially expressed during the spermatogenic cycle of seminiferous tubules, and Bmal1 and Clock deficient mice display significant CB morphological alterations due to BMAL1 ablation or low expression. These findings suggest that both BMAL1 and CLOCK contribute to CB assembly and physiology, raising questions on the role of the circadian clock in reproduction and on the molecular function that CLOCK and BMAL1 could potentially have in the CB assembly and physiology.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AP160P
    Nombre del producto:
    Rabbit Anti-Mouse IgG Antibody, HRP conjugate
  • Conversion of stereoisomers of leukotriene B4 to dihydro and tetrahydro metabolites by porcine leukocytes. 2160282

    We have previously shown that porcine leukocytes convert leukotriene B4 (LTB4) to two major products, 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4. Although we did not detect these products after incubation of LTB4 with human polymorphonuclear leukocytes, these cells converted 12-epi-6-trans-LTB4 to the corresponding 6,11-dihydro metabolite (i.e., there appeared to be a shift in the positions of the remaining double bonds). The objective of the present investigation was to determine whether 6-trans isomers of LTB4 are metabolized by porcine leukocytes by a pathway similar to LTB4, or whether they are metabolized by a pathway analogous to that in human leukocytes. We found that 6-trans-LTB4 and 12-epi-6-trans-LTB4 are metabolized more much extensively than LTB4 by porcine leukocytes. 6-trans-LTB4 appears to be converted by two different reductase pathways to two dihydro products differing in the positions of the two remaining double bonds between carbons 5 and 12. Dihydro-12-oxo and dihydro-5-oxo metabolites are also formed from this substrate. Porcine leukocytes also convert 6-trans-LTB4, presumably by a combination of the above two pathways, to tetrahydro, tetrahydro-12-oxo and tetrahydro-5-oxo metabolites, none of which possesses any conjugated double bonds. 12-epi-6-trans-LTB4 is also converted to tetrahydro metabolites by these cells. Experiments with deuterium-labeled 6-trans-LTB4 indicated that the deuterium in the 5-position was almost completely lost during the formation of tetrahydro-6-trans-LTB4, whereas about 80-85% of the deuterium in the 12-position was lost, suggesting a requirement for a 5-oxo intermediate. As with LTB4, 12-epi-8-cis-6-trans-LTB4, the product of the combined actions of 5-lipoxygenase and 12-lipoxygenase, was converted principally to dihydro and dihydro-12-oxo metabolites. Only a relatively small amount of the tetrahydro metabolite was detected.
    Tipo de documento:
    Referencia
    Referencia del producto:
    20-129
  • Use of benchtop exactive high resolution and high mass accuracy orbitrap mass spectrometer for screening in horse doping control. 21742125

    Liquid chromatography-mass spectrometry (LC-MS) has been widely used in doping control laboratories over the last two decades. Currently, simple quadrupole, triple quadrupole and ion trap are the most commonly employed analyzers in toxicological analysis. Nevertheless, the main lack of these technologies is the restricted number of target compounds simultaneously screened without loss of sensitivity. In this article we present an innovative screening approach routinely applied in the French horse doping control laboratory based on high resolution (50000) and high mass accuracy (<5 ppm) in full scan MS mode for more than 235 target analytes screened from an initial volume of 5 mL of urine. The sample preparation was classically founded on solid phase extraction by means of reverse phase C18 cartridges. LC-MS analyses were carried out on a Shimadzu binary HPLC pumps linked to a C18 Sunfire column associated with the high resolution exactive benchtop orbitrap mass spectrometer. This screening was performed alternatively in positive-negative ionization mode during the same run. Thus, the identification of compounds of interest was made using their exact mass in positive-negative ionization mode at their expected retention time. All data obtained were processed by ToxID software (ThermoFisherScientific) which is able to identify a molecule by theoretical mass and retention time. In order to illustrate this innovative technology applied in our laboratory, sample preparation, validation data performed on 20 target compounds from 16 different horse urine samples, chromatograms and spectra will be discussed in this paper.
    Tipo de documento:
    Referencia
    Referencia del producto:
    14-278
    Nombre del producto:
    Ras Assay Reagent (Raf-1 RBD, agarose)
  • LC/MS/MS structure elucidation of reaction intermediates formed during the TiO2 photocatalysis of microcystin-LR 18377943

    Microcystin-LR (MC-LR), a cyanotoxin and emerging drinking water contaminant, was treated with TiO2 photocatalysts immobilized on stainless steel plates as an alternative to nanoparticles in slurry. The reaction intermediates of MC-LR were identified with mass spectrometry (MS) at pH of Milli-Q water (pHsq = 5.7). Eleven new [M+H]+ were observed in the liquid chromatography mass spectrometry (LC/MS) chromatogram with some of them giving multiple peaks. Most of these reaction intermediates have not been reported from previous studies employing TiO2 nanoparticles at acidic conditions (pH = 4.0). Investigating the effects of pH (for 3.0
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo