ResourceLife Sciences
In-Cell Western™ Assay Cell Fixation/Permeabilisation Methods
9 Jun 2011To enable analysis of cellular proteins for the In-Cell Western (ICW) assay, cells must be immobilized onto a solid microplate surface, and antigens must be made accessible to their antibodies. In addition, permeabilisation is typically required to provide access to the antigen and subcellular localization. Here, two methods are presented for cell fixation and permeabilisation in ICW assays, one using methanol and another using formaldehyde/triton X-100. The methods are compared, and alternate methods are also suggested.
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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. Protein transfer can be performed by electroblotting with wet, semi-dry and dry transfer systems onto nitrocellulose and PVDF membranes. Blocking, washing and labeling of membranes follows, involving buffers, blocking reagents, blotting / incubation trays, labeling reagents, immunoblotting assays, antibodies and conjugates. Automated equipment for these steps is available to accelerate your lab workflow. Finally, detection and imaging of proteins can be conducted using gel documentation and imaging systems. Find the best western blotting equipment in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.Cell / Tissue CultureCell culture or tissue culture is used to study the biology of cells or tissues and to isolate cellular products in an environment which can be manipulated and well defined. Accurately control your culture environment with bioreactors or culture incubators, bind your cells to a surface or together with an extracellular matrix. Distinguish cell types with differential media or proliferate cells with certain characteristics using selective media. Enrich your media with supplements such as growth factors, sera and vitamins. Find the best cell and tissue culture products, kits and 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.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. :