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  • Comparison of standard capillary and chip separations of sodium dodecylsulfate–protein complexes 12685593

    Conditions for converting a set of five standard proteins to electrochemically active sodium dodecylsulfate (SDS) complexes were worked out with the aim of using such complexes for conductivity detection with a a chip electrophoresis system. The results obtained were compared with standard capillary electrophoresis (37 cm (effective length 30 cm)×75 μm I.D. capillary, 10 kV, negative polarity at the inlet). The chip separations were run at 500 V per chip (100 V/cm) as compared to the standard capillary arrangement, which was run at 266.6 V/cm. For the capillary set-up the protein complexes were prepared in aqueous solution (Milli-Q water) made 10 mM with respect to SDS. If the SDS concentration was increased to 50 mM, the separation in the capillary was incomplete. On the other hand with the chip system both approaches yielded acceptable results. The chip separations were slightly (but not distinctly) shorter and offered better separations than the standard set-up. The concentration of the surfactant used for the preparation the complexes results in alternations of the elution sequence, which is preserved if the chip separation is used instead of the capillary set-up. Apparently the full capacity of protein–SDS binding is not exploited for the preparation of the adducts.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere

  • Interactions of proteases, protease inhibitors, and the beta1 integrin/laminin gamma3 protein complex in the regulation of ectoplasmic specialization dynamics in the rat ... 14645107

    During spermatogenesis, developing germ cells migrate progressively across the seminiferous epithelium. This event requires extensive restructuring of cell-cell actin-based adherens junctions (AJs), such as the ectoplasmic specialization (ES, a testis-specific AJ type), between Sertoli cells and elongating/elongate spermatids. It was postulated that proteases and protease inhibitors worked in a yin-yang relationship to regulate these events. If this is true, then it is anticipated that both proteases and protease inhibitors are found at the ES. Indeed, matrix metalloprotease (MMP)-2, membrane-type 1 (MT1)-MMP and their inhibitor, tissue-inhibitor of metalloproteases (TIMP)-2, were shown to localize at the apical ES. In order to identify the putative MMP substrate as well as the unknown binding ligand for alpha6beta1 integrin in the ES, immunofluorescent microscopy coupled with immunoprecipitation techniques were used to demonstrate that laminin gamma3, largely a germ cell product, was present at the apical ES and could form a bona fide complex with beta1-integrin. Furthermore, the structural interactions of MMP-2 and MT1-MMP with laminin gamma3 and beta1-integrin, but not with N-cadherin or nectin-3, have implicated the crucial role of MMP-2/MT1-MMP in the regulation of integrin/laminin-based ES dynamics. Using an in vivo model to study AJ dynamics where adult rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) to disrupt Sertoli-germ cell adhesive function, an induction of active MMP-2, active MT1-MMP and TIMP-2 but not active MMP-9 was detected between 0.5 and 8 h after AF-2364 treatment. This time frame coincided with the depletion of elongating/elongate spermatids from the epithelium, illustrating the synergistic relationships between MMP-2, MT1-MMP, and TIMP-2 in AJ disassembly. Perhaps the most important of all, the use of a specific MMP-2 and MMP-9 inhibitor, (2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid, could effectively delay the AF-2364-induced elongating/elongate spermatid loss from the epithelium, demonstrating the pivotal role of MMP-2 activation in ES disassembly. Collectively, these studies illustrate that the beta1-integrin/laminin gamma3 complex is a putative ES-structural protein complex, which is regulated, at least in part, by the activation of MMP-2 involving MT1-MMP and TIMP-2 at the apical ES. The net result of this interaction likely regulates germ cell movement in the seminiferous epithelium.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Pregnant women exercise at a higher intensity during 30 min of self-paced cycling compared with walking during late gestation: implications for 2 h postprandial glucose l ... 23332446

    To compare the effect of an acute 30-min bout of self-paced stationary cycling (SC) with treadmill walking (TW) or a resting control (CON) on maternal blood glucose, insulin and metabolic responses during pregnancy.Twelve healthy women at 29.9±2.4 (mean±SD) weeks gestation consumed a 75 g carbohydrate drink as part of an oral glucose tolerance test (OGTT) following 30 min of SC, TW or CON. Blood was sampled before and after exercise, and for 2 h in response to the OGTT for the determination of glucose and insulin. Exercise intensity was monitored and enjoyment of TW and SC was assessed post-exercise.Women selectively worked harder in SC compared with TW, with a higher maternal heart rate, rating of perceived exertion, mean oxygen consumption, respiratory exchange ratio, and total energy expenditure during exercise (p<0.05). SC was also associated with significantly lower postprandial blood glucose levels at 120 min following the OGTT (6.9±0.9 mmoll(-1)) compared with both CON (8.1±0.7 mmoll(-1), p=0.001) and TW (7.8±0.9 mmoll(-1), p=0.004) and lower insulin at 120 min post-glucose ingestion compared with TW (p=0.021). Enjoyment was similar between exercise protocols (p=0.437).In late pregnancy, an acute 30 min bout of self-paced SC may be preferable to a matched duration of TW given the additional energy expenditure that can be achieved, which in turn appears beneficial for blunting the glycemic response to glucose ingestion.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    HMHMAG-34K

  • Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies. 21925951

    Zaire ebolavirus (ZEBOV) can be transmitted by human-to-human contact and causes acute haemorrhagic fever with case fatality rates up to 90%. There are no effective therapeutic or prophylactic treatments available. The sole transmembrane glycoprotein (GP) is the key target for developing neutralizing antibodies. In this study, recombinant VSVΔG/ZEBOVGP was used to generate monoclonal antibodies (MAbs) against the ZEBOV GP. A total of 8 MAbs were produced using traditional hybridoma cell fusion technology, and then characterized by ELISA using ZEBOV VLPs, Western blotting, an immunofluorescence assay, and immunoprecipitation. All 8 MAbs worked in IFA and IP, suggesting that they are all conformational MAbs, however six of them recognized linearized epitopes by WB. ELISA results demonstrated that one MAb bound to a secreted GP (sGP 1-295aa); three bind to a part of the mucin domain (333-458aa); three MAbs recognized epitopes on the C-terminal domain of GP1 (296-501aa); and one bound to full length GP (VLPs/GP1,2 ΔTm). Using a mouse model these MAbs were evaluated for their therapeutic capacity during a lethal infection. All 8 MAb improved survival rates by 33%-100% against a high dose lethal challenge with mouse-adapted ZEBOV. This work has important implications for further development of vaccines and immunotherapies for ZEBOV infection.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Clonal growth of normal human epidermal keratinocytes in a defined medium. 7040427

    Colony formation by normal human epidermal keratinocytes (HK) has been achieved in a medium that contains no deliberately added undefined supplements. The term "defined" is used to describe this medium, although the possibility that trace contaminants in its components could be contributing to the multiplication that it supports cannot yet be ruled out completely. The defined medium consists of a basal medium, MCDB 152, supplemented with 5 ng/ml epidermal growth factor (EGF), 10 micrograms/ml transferrin, 5 micrograms/ml insulin, 1.4 X 10(-6) M hydrocortisone, 1.0 X 10(-5) Methanolamine, 1.0 X 10(-5) M phosphoethanolamine, and 2.0 X 10(-9) M progesterone. MCDB 152 differs from MCDB 151, previously developed for multiplication of HK with small amounts of dialyzed serum (Peehl and Ham, 1980b), only by addition of the trace element mixture from human fibroblast medium MCDB 104 (McKeehan et al., 1977). Most of the requirement for transferrin, which is the least defined component of the defined medium, can be replaced by adding freshly dissolved and sterilized ferrous sulfate to the final medium after it has been filter sterilized. Insulin and EGF are clearly needed for optimal multiplication and hydrocortisone is mildly beneficial. Either ethanolamine or phosphoethanolamine must be present in the defined medium for HK multiplication. There is a greater need for EGF and less for hydrocortisone in the defined medium than in previous partially defined systems that we have worked with. Very large colonies of flattened epithelial cells are obtained in the defined medium, which has a low calcium concentration (0.03 mM) and does not favor keratinocyte differentiation. Less growth and more differentiation are obtained with higher calcium concentrations. The defined medium is highly selective for keratinocyte growth from a mixed inoculum of keratinocytes and fibroblasts.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • cDNA cloning of mouse tumor necrosis factor-alpha converting enzyme (TACE) and partial analysis of its promoter. 10375622

    Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces pleiotropic inflammatory reactions. Soluble TNFalpha is released from its membrane-bound precursor by TNFalpha converting enzyme (TACE)/a disintegrin and metalloproteinase 17 (ADAM17). We have recently cloned the mouse TACE complete cDNA and the 5' flanking promoter region of the gene. Two versions of the mouse TACE cDNA having a coding region of 2541bp were obtained: one was about 4.1kb and the other was 4.4kb in length, in which only the length of the 3' UTR was different. Rapid amplification of 5' cDNA ends suggested that there were multiple transcriptional start sites. From the coding sequence, 827 amino acids were deduced which were 91.9 % identical to those of human TACE. Northern blot analysis indicated that the major transcript was approx. 4.4kb product in the mouse. The mouse TACE mRNA was ubiquitously expressed, and was particularly high in the lung. The proximal promoter contained multiple AP2 and Sp1 transcription factor binding sites and included a GC box and a CCAAT box, but lacked a consensus TATA box. Reporter gene analysis using RAW264.7 cells showed that the fragment at nt -290 to -1 from the translation start site has a strong promoter activity, and appeared to be essential for transcription of the mouse TACE mRNA. Finally, we found that the mouse TACE promoter at nt -2304 to -1 also worked in NIH3T3, 3T3L1 and C6 cell lines.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    AB19027
    Produktbezeichnung:
    Anti-ADAM 17 Antibody

  • Distinct roles of neuroligin-1 and SynCAM1 in synapse formation and function in primary hippocampal neuronal cultures. 22542674

    Neuroligins are a family of cell adhesion molecules critical in establishing proper central nervous system connectivity; disruption of neuroligin signaling in vivo precipitates a broad range of cognitive deficits. Despite considerable recent progress, the specific synaptic function of neuroligin-1 (NL1) remains unclear. A current model proposes that NL1 acts exclusively to mature pre-existent synaptic connections in an activity-dependent manner. A second element of this activity-dependent maturation model is that an alternate molecule acts upstream of NL1 to initiate synaptic connections. SynCAM1 (SC1) is hypothesized to function in this capacity, though several uncertainties remain regarding SC1 function. Using overexpression and chronic pharmacological blockade of synaptic activity, we now demonstrate that NL1 is capable of robustly recruiting synapsin-positive terminals independent of synaptic maturation and activity in 2-week old primary hippocampal neuronal cultures. We further report that neither SC1 overexpression nor knockdown of endogenous SC1 impacts synapsin punctum densities, suggesting that SC1 is not a limiting factor of synapse initiation in maturing hippocampal neurons in vitro. Consistent with these findings, we observed profoundly greater recruitment of synapsin-positive presynaptic terminals by NL1 than SC1 in a mixed-culture assay of artificial synaptogenesis between primary neurons and heterologous cells. Collectively, our results contend multiple aspects of the proposed model of NL1 and SC1 function and motivate an alternative model whereby SC1 may mature synaptic connections forged by NL1. Supporting this model, we present evidence that combined NL1 and SC1 overexpression triggers excitotoxic neurodegeneration through SC1 signaling at synaptic connections initiated by NL1.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    AB1543
    Produktbezeichnung:
    Anti-Synapsin I Antibody

  • Networked T cell death following macrophage infection by Mycobacterium tuberculosis. 22675566

    Depletion of T cells following infection by Mycobacterium tuberculosis (Mtb) impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised.We found that lymphopenia (less than 1.5 × 10(9) cells/l) was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb) or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s) were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG)- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system.Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as interfere with microbial eradication in the granuloma.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    05-338
    Produktbezeichnung:
    Anti-Fas Antibody (human, neutralizing), clone ZB4

  • Profiling post-transcriptionally networked mRNA subsets using RIP-Chip and RIP-Seq. 24257445

    Post-transcriptional regulation of messenger RNA contributes to numerous aspects of gene expression. The key component to this level of regulation is the interaction of RNA-binding proteins (RBPs) and their associated target mRNA. Splicing, stability, localization, translational efficiency, and alternate codon use are just some of the post-transcriptional processes regulated by RBPs. Central to our understanding of these processes is the need to characterize the network of RBP-mRNA associations and create a map of this functional post-transcriptional regulatory system. Here we provide a detailed methodology for mRNA isolation using RBP immunoprecipitation (RIP) as a primary partitioning approach followed by microarray (Chip) or next generation sequencing (NGS) analysis. We do this by using specific antibodies to target RBPs for the capture of associated RNA cargo. RIP-Chip/Seq has proven to be is a versatile, genomic technique that has been widely used to study endogenous RBP-RNA associations.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • The interaction of force and repetition on musculoskeletal and neural tissue responses and sensorimotor behavior in a rat model of work-related musculoskeletal disorders. 24156755

    We examined the relationship of musculoskeletal risk factors underlying force and repetition on tissue responses in an operant rat model of repetitive reaching and pulling, and if force x repetition interactions were present, indicative of a fatigue failure process. We examined exposure-dependent changes in biochemical, morphological and sensorimotor responses occurring with repeated performance of a handle-pulling task for 12 weeks at one of four repetition and force levels: 1) low repetition with low force, 2) high repetition with low force, 3) low repetition with high force, and 4) high repetition with high force (HRHF).Rats underwent initial training for 4-6 weeks, and then performed one of the tasks for 12 weeks, 2 hours/day, 3 days/week. Reflexive grip strength and sensitivity to touch were assayed as functional outcomes. Flexor digitorum muscles and tendons, forelimb bones, and serum were assayed using ELISA for indicators of inflammation, tissue stress and repair, and bone turnover. Histomorphometry was used to assay macrophage infiltration of tissues, spinal cord substance P changes, and tissue adaptative or degradative changes. MicroCT was used to assay bones for changes in bone quality.Several force x repetition interactions were observed for: muscle IL-1alpha and bone IL-1beta; serum TNFalpha, IL-1alpha, and IL-1beta; muscle HSP72, a tissue stress and repair protein; histomorphological evidence of tendon and cartilage degradation; serum biomarkers of bone degradation (CTXI) and bone formation (osteocalcin); and morphological evidence of bone adaptation versus resorption. In most cases, performance of the HRHF task induced the greatest tissue degenerative changes, while performance of moderate level tasks induced bone adaptation and a suggestion of muscle adaptation. Both high force tasks induced median nerve macrophage infiltration, spinal cord sensitization (increased substance P), grip strength declines and forepaw mechanical allodynia by task week 12.Although not consistent in all tissues, we found several significant interactions between the critical musculoskeletal risk factors of force and repetition, consistent with a fatigue failure process in musculoskeletal tissues. Prolonged performance of HRHF tasks exhibited significantly increased risk for musculoskeletal disorders, while performance of moderate level tasks exhibited adaptation to task demands.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere