ResourceLife Sciences
Simultaneous Chemiluminescent and Fluorescent Detection: A Novel Approach for Double Western Blot Analysis of Low Copy Protein Targets
9 May 2011In this application note by Carestream Molecular Imaging, the effects of the addition of HRP substrates, on the fluorescent signal of Alexa Fluor® labelled antibodies in Western blotting are assessed. Additionally, a protocol is presented that describes both the capture of luminescent and fluorescent images, as well as post image capture processing (i.e. pseudocolor and image overlay) to produce publication quality figures. Chemiluminescent Western blot analysis remains the most sensitive method for detection of low-copy protein targets. Since most loading control proteins are highly expressed proteins that can be easily detected using fluorescence, and most researchers are trying to detect low-copy proteins of interest, a method is developed for simultaneous incubation of two protein targets using both fluorescent and chemiluminescent secondary antibodies.
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ElectrophoresisElectrophoresis equipment is used to separate mixtures of protein, DNA or RNA, based on their electric charge, size and other physical characteristics, by passing them through a medium such as a polyacrylamide gel, an agarose gel or a capillary tube. Electrophoresis equipment includes horizontal and vertical gel electrophoresis chambers, isoelectric focusing systems, 2D electrophoresis and capillary electrophoresis instruments. Precast gels with a gradient can be used or gels can be hand cast. Samples are run alongside ladders or markers to identify the approximate size of a molecule. Separated proteins and nucleic acids in the gel can be stained or probed with fluorescent markers and then imaged and detected using gel documentation instruments, transilluminators, densitometers and scanners. Find the best electrophoresis equipment in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.AntibodiesAntibodies are used in techniques such as confocal and fluorescence microscopy, flow cytometry, ELISA, ELISPOT, immunohistochemistry, western blotting and immunopreciptation. Select specific antigen reactivity, high specific affinity, low non-specific binding, monoclonal or polyclonal, primary or secondary antibodies and associated conjugates such as an enzyme or dye for visualization.Western BlottingWestern blotting equipment is used to transfer and identify specific proteins within a sample, reveal protein modifications, as well as give a semi-quantitative estimation of their concentration. Western blotting equipment includes all apparatus necessary to transfer proteins from gel to membrane and subsequent processing steps. 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Find the best PCR kits and thermal cyclers and purification equipment in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.Cell Lines Stem Cells and Primary CellsPrimary cell cultures, established cell lines and stem cells are vital for <i>in vitro</i> and <i>ex vivo</i> experimentation. High-quality cells, optimized for your applications, alongside optimized cell substrates, growth medium and supplements, are critical for experimental success. Explore a range of cells suitable for your applications, including isogenic cell lines, competent cells, induced pluripotent stem cell (iPSC)-derived cell lines, fungal/bacterial/mammalian cell lines, stem cells and cancer cell lines. Find the best cells for your research in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.Gel Doc / Image AnalysisGel documentation (gel doc) or gel imaging systems are used for the analysis of proteins, antibodies and nucleic acid immobilized in polyacrylamide or agarose gels, membranes or microarrays. Explore a range of a gel imaging systems, densitometers, scanners, transilluminators or UV lamp + CCD cameras for your image analysis solutions. Colorimetric, fluorescent and/or radioisotopic samples can be visualized and documented for further analysis. See gel doc / Image analysis software for quantitative 1D and 2D analysis of your samples. Find the best gel doc / image analysis products in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.Cell-Based AssaysCell-based assays are used to monitor the presence, quantity and activities of a desired cellular analyte including drug molecules or biomarkers. This can reveal information on cell health (apoptosis, cytotoxicity, viability and proliferation assays), cell metabolism, cell migration and cell signaling mechanisms. Find the best cell-based assay products, kits and equipment with our peer reviewed product directory: compare products, check customer reviews and receiving pricing direct from manufacturers.Protein PurificationProtein purification is a vital step in drug discovery, therapeutics, biotech and life science research. The purification process typically involves subcellular or membrane protein extraction with cell lysis kits, separation of proteins from cell debris by filtration or spin columns, and the isolation of proteins of interest from other proteins and impurities with affinity purification (including fusion protein tags and antibody binding proteins A, G and L), immunoprecipitation or chromatographic methods, such as ion exchange, size exclusion and immobilized metal affinity chromatography. All purification methods come in multiple formats for your laboratory needs, including agarose or magnetic beads, resins, columns and filter plates. Find the best protein purification equipment in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.Gene Expression and Molecular CloningMolecular cloning is a set of techniques that utilizes vectors to transfer recombinant DNA into host cells and is an essential tool for investigating the expression of genes and proteins in bacterial or mammalian cells. A variety of vectors optimized for gene cloning and expression in a range of host organisms are available, alongside competent cells for genetic replication. Here, you can explore a range of molecular tools, high-quality genomic and cDNA libraries, premade clones, transformation and transfection reagents and mutagenesis or gene expression detection assays and expression arrays. Find the best gene expression and molecular cloning products in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.BiomarkersBiomarkers are biological markers which can be measured and evaluated to indicate a biological state. The use of biomarkers in research and diagnosis can indicate a normal or disease state or drug response of cells / tissues. Biomarkers include genetic markers, cell surface markers such as antigens, antibodies or receptors and secreted molecules such as cytokines. An assay system is required for identification of biomarkers. :