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  • Prion protein oligomers in Creutzfeldt-Jakob disease detected by gel-filtration centrifuge columns. 19389076

    Prion diseases are diagnosed by the detection of accumulation of abnormal prion protein (PrP) using immunohistochemistry or the detection of protease-resistant abnormal PrP (PrP(res)). Although the abnormal PrP is neurotoxic by forming aggregates, recent studies suggest that the most infectious units are smaller than the amyloid fibrils. In the present study, we developed a simplified method by applying size-exclusion gel-filtration chromatography to examine PrP oligomers without proteinase K digestion in Creutzfeldt-Jakob disease (CJD) samples, and evaluated the correlation between disease severity and the polymerization degree of PrP. Brain homogenates of human CJD and non-CJD cases were applied to the gel-filtration spin columns, and fractionated PrP molecules in each fraction were detected by western blot. We observed that PrP oligomers could be detected by the simple gel-filtration method and distinctly separated from monomeric cellular PrP (PrP(c)). PrP oligomers were increased according to the disease severity, accompanied by the depletion of PrP(c). The separated PrP oligomers were already protease-resistant in the case with short disease duration. In the cases with quite severe pathology the oligomeric PrP reached a plateau, which may indicate that PrP molecules could mostly develop into amyloid fibrils in the advanced stages. The increase of PrP oligomers correlated with the degree of histopathological changes such as spongiosis and gliosis. The decrease of monomeric PrP(c) was unexpectedly obvious in the diseased cases. Dynamic changes of both oligomerization of the human PrP and depletion of normal PrP(c) require further elucidation to develop a greater understanding of the pathogenesis of human prion diseases.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    AP192P
    Produktbezeichnung:
    Donkey Anti-Mouse IgG Antibody, HRP conjugate, Species Adsorbed

  • A protein profile study to discriminate CIN lesions from normal cervical epithelium. 21573931

    Cervical intraepithelial neoplasia (CIN), a frequently encountered disease caused by Human Papilloma Virus (HPV) is often diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. Since it is known that this procedure strongly affects the water-soluble proteins contained in the cervical tissue we decided to investigate whether a water-soluble protein-saving biopsy processing method can be used to support the diagnosis of normal and CIN.Cervical punch biopsies from 55 women were incubated for 24 h at 4°C in RPMI1640 medium for protein analysis prior to usual FFPE processing and p16 and Ki67-supported histologic consensus diagnosis was assessed. The biopsy supernatants were subjected to surface-enhanced laser desorption-ionization time of flight mass spectrometry (SELDI-TOF MS) for identifying differentially expressed proteins. Binary logistic regression and classification and regression trees (CART) were used to develop a classification model.The age of the patients ranged from 26 to 40 years (median 29.7). The consensus diagnoses were normal cervical tissue (n = 10) and CIN2-3 (n = 45). The mean protein concentration was 1.00 and 1.09 mg/ml in the normal and CIN2-3 group, respectively. The peak detection and clustering process resulted in 40 protein peaks. Many of these peaks differed between the two groups, but only three had independent discriminating power. The overall classification results were 88%.Water-soluble proteins sampled from punch biopsies are promising to assist the diagnosis of normal and CIN2-3.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Protein kinase D1 mediates stimulation of DNA synthesis and proliferation in intestinal epithelial IEC-18 cells and in mouse intestinal crypts. 21051537

    We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p less than 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p less than 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    04-787

  • Detection of protein alterations in male breast cancer using two dimensional gel electrophoresis and mass spectrometry: the involvement of several pathways in tumorigenes ... 17996735

    BACKGROUND: Little emphasis has been placed today on the elucidation of protein alterations in male breast carcinogenesis. METHODS: Protein extracts were subjected to both isoelectric focusing (IEF) and non-equilibrium pH gradient electrophoretic (NEPHGE) analyses. Differentially expressed proteins in tumor tissues were identified by matrix assisted laser desorption /ionization time of flight (MALDI-TOF) mass spectrometry and database search. RESULTS: Some of the alterations involve variations in the expression of cytokeratins 8, 18 and 19. More interestingly, tropomyosin1, a protein known to play a role in suppression of the malignant phenotype, was found to be under-expressed in cancer tissues, implicating a possible pivotal role for this protein in male breast carcinogenesis. Co-upregulation of molecular chaperones (heat shock protein HSP27 and protein disulfide isomerase), stress related proteins (peroxiredoxin 1 and peptidylprolyl isomerase A) and glycolytic enzymes (enolase 1) occurred also in male breast tumors. Some of the remaining alterations include proteins involved in invasion and metastasis, such as galectin 1 and cathepsin D. CONCLUSIONS: The present study represents a first proteomic investigation of protein alterations in infiltrating ductal carcinomas (IDCA) of the male breast. A number of protein alterations in tumor tissues have been characterised thus, providing new insights into the molecular mechanisms underlying this disease.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    AB5449
    Produktbezeichnung:
    Anti-Tropomyosin 4 Antibody

  • Prion protein detection using nanomechanical resonator arrays and secondary mass labeling. 18271602

    Nanomechanical resonators have shown potential application for mass sensing and have been used to detect a variety of biomolecules. In this study, a dynamic resonance-based technique was used to detect prion proteins (PrP), which in conformationally altered forms are known to cause neurodegenerative diseases in animals as well as humans. Antibodies and nanoparticles were used as mass labels to increase the mass shift and thus amplify the frequency shift signal used in PrP detection. A sandwich assay was used to immobilize PrP between two monoclonal antibodies, one of which was conjugated to the resonator's surface while the other was either used alone or linked to the nanoparticles as a mass label. Without additional mass labeling, PrP was not detected at concentrations below 20 microg/mL. In the presence of secondary antibodies the analytical sensitivity was improved to 2 microg/mL. With the use of functionalized nanoparticles, the sensitivity improved an additional 3 orders of magnitude to 2 ng/mL.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    17-191
    Produktbezeichnung:
    MAP Kinase/Erk Assay Kit, non-radioactive

  • Small heat shock protein 27 (Hsp27) expression is highly induced in rat myometrium during late pregnancy and labour. 15615903

    The underlying mechanisms that regulate uterine contractions during labour are still poorly understood. A candidate regulatory protein is heat shock protein 27 (Hsp27). It belongs to the small heat shock protein family and can regulate actin cytoskeleton dynamics, act as a chaperone, and may regulate contractile protein activation. As a result, we hypothesized that Hsp27 expression would be highly induced during late pregnancy and labour. Hsp27 mRNA expression was significantly elevated (P less than 0.05) on days 17 to 22 of gestation. In addition, immunoblot analysis demonstrated that detection of total Hsp27 increased (P less than 0.05) between day 21 and 1 day post-partum (PP) inclusive. Since phosphorylation of Hsp27 has been reported to be a prerequisite for smooth muscle contraction, we examined the temporal and spatial expression of Ser-15 phosphorylated Hsp27. Immunoblot analysis showed that the detection of Ser-15 phosphorylated Hsp27 significantly increased (P less than 0.05) between days 19 and 23 (active labour) inclusive, in parallel with detection of total Hsp27. Immunocytochemical analysis of Ser-15 phosphorylated Hsp27 expression in situ demonstrated that phosphorylated Hsp27 in circular muscle became detectable in peri-nuclear and membrane regions on days 19 to 22, but was primarily restricted to the cytoplasm on days 23 to PP. In contrast, phosphorylated Hsp27 in longitudinal muscle was primarily detected in myocyte membranes on days 15 to 22, and then also became detectable in the cytoplasm of myocytes on days 23 and PP. Our results demonstrate that Hsp27 expression is highly upregulated during late pregnancy and labour and suggest that Hsp27 is a potential candidate contraction-associated protein.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    06-517

  • Surfactant protein B detection and gene expression in chronic rhinosinusitis. 17507829

    Surfactant protein (SP)-B is a hydrophobic protein secreted within pulmonary surfactant that facilitates the adsorption of surface-active lipids to the air-liquid interface of the alveoli and increases alveolar stability. SP-B may also have anti-inflammatory properties. It is implicated in decreasing the pulmonary inflammatory response to bacterial lipopolysaccharide. However, the expression and function of SP-B in the sinonasal cavities has not been elucidated. Our objective was to detect the presence of SP-B, measure alterations in several forms of chronic rhinosinusitis (CRS), and localize cellular protein expression.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    MAB3278

  • Utility of surfactant protein B precursor and thyroid transcription factor 1 in differentiating adenocarcinoma of the lung from malignant mesothelioma. 10374779

    Differentiation of malignant mesothelioma from adenocarcinoma, particularly from a lung primary, remains a difficult diagnostic problem. Surfactant protein B precursor (pro-SP-B) and thyroid transcription factor 1 (ITF-1) are expressed selectively in the normal respiratory epithelium and in adenocarcinomas of the lung. In this study, we evaluated the utility of pro-SP-B and ITF-1 in distinguishing pulmonary adenocarcinomas and malignant mesotheliomas. Immunoreactivity for pro-SP-B and TTF-1 was examined in paraffin sections of 370 primary lung carcinomas (208 adenocarcinomas, 101 squamous cell carcinomas, and 61 large cell carcinomas) and 95 malignant mesotheliomas, using a pro-SP-B antiserum and a monoclonal TTF-1 antibody with a biotin-streptavidin detection system. Immunostaining for pro-SP-B was detected in 57% of adenocarcinomas, and 20% of large cell carcinomas. Immunoreactivity for TTF-1 was shown in 76% of adenocarcinomas and 26% of large cell carcinomas. Malignant mesotheliomas and squamous cell carcinomas did not stain with either antibody. The expression of pro-SP-B and TTF-1 in adenocarcinomas of the lung but not in malignant mesotheliomas shows that pro-SP-B and TTF-1 staining is useful in differentiating these neoplasms.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    07-601
    Produktbezeichnung:
    Anti-TTF-1 Antibody