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  • Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum. 21806555

    The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Multiplex immunoassays of peptide hormones extracted from formalin-fixed, paraffin-embedded tissue accurately subclassify pituitary adenomas. 22205691

    The current gold standard for diagnostic classification of many solid-tissue neoplasms is immunohistochemistry (IHC) performed on formalin-fixed, paraffin-embedded (FFPE) tissue. Although IHC is commonly used, there remain important issues related to preanalytic variability, nonstandard methods, and operator bias that may contribute to clinically significant error. To increase the quantitative accuracy and reliability of FFPE tissue-based diagnosis, we sought to develop a clinical proteomic method to characterize protein expression in pathologic tissue samples rapidly and quantitatively.We subclassified FFPE tissue from 136 clinical pituitary adenoma samples according to hormone translation with IHC and then extracted tissue proteins and quantified pituitary hormones with multiplex bead-based immunoassays. Hormone concentrations were normalized and compared across diagnostic groups. We developed a quantitative classification scheme for pituitary adenomas on archived samples and validated it on prospectively collected clinical samples.The most abundant relative hormone concentrations differentiated sensitively and specifically between IHC-classified hormone-expressing adenoma types, correctly predicting IHC-positive diagnoses in 85% of cases overall, with discrepancies found only in cases of clinically nonfunctioning adenomas. Several adenomas with clinically relevant hormone-expressing phenotypes were identified with this assay yet called "null" by IHC, suggesting that multiplex immunoassays may be more sensitive than IHC for detecting clinically meaningful protein expression.Multiplex immunoassays performed on FFPE tissue extracts can provide diagnostically relevant information and may exceed the performance of IHC in classifying some pituitary neoplasms. This technique is simple, largely amenable to automation, and likely applicable to other diagnostic problems in molecular pathology.
    Document Type:
    Reference
    Product Catalog Number:
    AB928
    Product Catalog Name:
    Anti-Follicle Stimulating Hormone Antibody
  • Detection of natural peptide antibiotics in human nasolacrimal ducts. 11527925

    PURPOSE: To determine the expression and production of antimicrobial peptides by mucosal cells of the lacrimal passage in healthy and pathologic states. METHODS: Detection of bactericidal-permeability-increasing protein (BPI), heparin-binding protein (CAP37), human cationic antimicrobial protein (LL-37), human alpha-defensin 5 (HD5), human alpha-defensin 6 (HD6), human beta-defensin 1 (HBD-1), and human beta-defensin 2 (HBD-2) was performed by reverse transcription-polymerase chain reaction (RT-PCR). Intracellular deposition of lysozyme, lactoferrin, secretory phospholipase A(2), human neutrophil defensins (HNP-1, -2, and -3), human beta-defensin 1 (HBD-1), and human beta-defensin 2 (HBD-2) was analyzed immunohistochemically. Samples were obtained from 15 patients by surgery and from 10 cadavers. RESULTS: RT-PCR revealed BPI, CAP37, and HBD-1 mRNA in samples of healthy nasolacrimal duct epithelium. Additionally, HBD-2 mRNA was detected in epithelial samples from patients with dacryocystitis. Messenger RNAs for LL-37 and alpha-defensin 5 and 6 were absent in all samples investigated. Immunohistochemistry revealed lysozyme, lactoferrin, secretory phospholipase A(2), and HNP-1, -2, and -3 to be present in all samples, whereas HBD-1 was present only in some of the healthy and inflamed samples. Immunoreactive HBD-2 peptide was visible only in some of the inflamed samples. CONCLUSIONS: The data suggest that the human efferent tear ducts produce a broad spectrum of antimicrobial peptides. Under inflammatory conditions, changes in the expression pattern occurred, revealing induction of the human inducible defensin HBD-2 and in some cases downregulation of HBD-1 and CAP37. Antimicrobial peptides have a therapeutic potential in dacryocystitis, in that they have a broad spectrum of antimicrobial activity and accelerate epithelial healing. However, caution is appropriate, because defensins also promote fibrin formation and cell proliferation, which are key elements in scarring processes, such as dacryostenosis.
    Document Type:
    Reference
    Product Catalog Number:
    05-143
    Product Catalog Name:
    Anti-PLA2 Antibody, secretory
  • Detection of ubiquitinated huntingtin species in intracellular aggregates. 25674046

    Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins. Huntington's disease (HD) is one of nine diseases caused by an expanded polyQ repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin (Htt) fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated Htt such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated Htt fragments offers an important possibility to understand Htt degradation and aggregation processes within the cell. For the identification of aggregated Htt and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant Htt. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1574
    Product Catalog Name:
    Anti-Polyglutamine-Expansion Diseases Marker Antibody, clone 5TF1-1C2