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  • Evaluation of seven oestrogen receptor beta antibodies for immunohistochemistry, western blotting, and flow cytometry in human breast tissue. 12015738

    Two oestrogen receptors, ER alpha and ER beta, exist. While much is known about ER alpha, the role of ER beta is still undefined, especially at the protein level. The aim of this study was to determine the utility of seven ER beta antibodies (14C8, 8D5, PAI313, PPG5/10, N19, 9.88, and D7N) raised against different domains of ER beta in three commonly used laboratory applications, namely immunohistochemistry, western blot, and flow cytometry, using human breast material. For immunohistochemical analysis of frozen material, PAI313 and D7N gave stronger and more specific signals than 14C8, 8D5, and PPG5/10. In paraffin sections, 14C8, closely followed by PPG5/10, gave by far the most superior nuclear immunoreactivity, compared with the other antibodies tested. In general, flow cytometry results mirrored the immunohistochemistry data for paraffin sections, with antibodies ranked 14C8 > 8D5> or = PAI-313 > PPG5/10 >D7N. For western blotting, 8D5 and D7N yielded the strongest and most consistent bands, with weaker bands seen with the others. It is concluded that ER beta protein can be detected using specific antibodies. However, there is considerable variation between the specificity and application of these antibodies, highlighting the fact that careful optimization is required when selecting an antibody for use in a particular laboratory technique.
    Document Type:
    Reference
    Product Catalog Number:
    06-629

  • Detecting protein-protein interactions by Far western blotting. 18079728

    Far western blotting (WB) was derived from the standard WB method to detect protein-protein interactions in vitro. In Far WB, proteins in a cell lysate containing prey proteins are firstly separated by SDS or native PAGE, and transferred to a membrane, as in a standard WB. The proteins in the membrane are then denatured and renatured. The membrane is then blocked and probed, usually with purified bait protein(s). The bait proteins are detected on spots in the membrane where a prey protein is located if the bait proteins and the prey protein together form a complex. Compared with other biochemical binding assays, Far WB allows prey proteins to be endogenously expressed without purification. Unlike most methods using cell lysates (e.g., co-immunoprecipitation (co-IP)) or living cells (e.g., fluorescent resonance energy transfer (FRET)), Far WB determines whether two proteins bind to each other directly. Furthermore, in cases where they bind to each other indirectly, Far WB allows the examination of candidate protein(s) that form a complex between them. Typically, 2-3 d are required to carry out the experiment.
    Document Type:
    Reference
    Product Catalog Number:
    AQ132P
    Product Catalog Name:
    Goat Anti-Rabbit IgG Antibody, F(ab')2, HRP conjugate

  • New monoclonal antibodies to mesothelin useful for immunohistochemistry, fluorescence-activated cell sorting, Western blotting, and ELISA. 16115924

    Mesothelin is a cell surface protein that is highly expressed in some malignant tumors, and is a promising target for immunotherapy. Recent data suggests that mesothelin is an adhesive protein and may have a role in the metastases of ovarian cancer. Although a few monoclonal antibodies (MAb) to mesothelin have been produced, they have limitations for the study of expression of native mesothelin because of their low affinity or reactivity only with denatured mesothelin protein. We have produced novel MAbs to mesothelin to help study mesothelin function and to develop improved diagnosis and immunotherapy of mesothelin-expressing tumors.Mesothelin-deficient mice were immunized with plasmid cDNA encoding mesothelin, and boosted with a mesothelin-rabbit IgG Fc fusion protein prior to cell fusion. Hybridomas were screened by an ELISA using plates coated with mesothelin-Fc protein.Seventeen hybridomas producing anti-mesothelin antibodies were established and shown to react with two epitopes on mesothelin. One group reacts with the same epitope as the low affinity antibody K1 that was originally used to identify mesothelin. The other is a new group that reacts with a new epitope. One antibody from each group was chosen for further study and shown to react strongly on ELISA, on immunohistochemistry, and by fluorescence-activated cell sorting on living cells.Our two newly established MAbs, MN and MB, have different and useful properties compared with current antibodies used for the detection of mesothelin by immunohistochemistry, fluorescence-activated cell sorting, ELISA, and Western blotting.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. 24023619

    Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.
    Document Type:
    Reference
    Product Catalog Number:
    AB9568
    Product Catalog Name:
    Anti-Neurofilament L Antibody