The use of total protein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting.
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Tandem scanning reflected-light microscopy of cell-substratum adhesions and stress fibres in Swiss 3T3 cells.
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This paper describes two applications of the tandem scanning reflected-light microscope (TSM) for the observation of the structure of individual cells growing in tissue culture. First, the TSM is used as an alternative to interference reflection microscopy (IRM) or total internal reflection aqueous fluorescence microscopy (TIRAF) to observe cell-substratum adhesions in unstained living cells growing on a glass coverslip. Second, the TSM is used to produce improved images of cellular structures in 3T3 cells stained with various protein dyes including Napthol Blue Black (NBB) and Coomassie Brilliant Blue (CBB). More specifically, close contacts and focal contacts are resolved in living 3T3 cells, and features of the nucleus, the cytoskeleton and extracellular matrix are resolved in both NBB- and CBB-stained cells. The focal contacts and associated stress fibres are clearly imaged in NBB-stained cells. The TSM is an improvement over conventional incident light microscopy because of the confocal image excludes information from out-of-focus regions of the cytoplasm, and, unlike the laser-based confocal microscope, the actual colour of the specimen is viewed directly with TSM in almost real-time. (+info)
Remazol Brilliant Blue as a pre-stain for the immediate visualization of human serum proteins on polyacrylamide gel disc electrophoresis.
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We describe the pre-staining of proteins in normal human serum with Remazol Brilliant Blue before separation by disc electrophoresis. Critical to reproducibility are: dye concentration of 0.16 mol/L in a Tris-glycine buffer (pH 8.3), use of equal volumes of serum and dye solution, a tagging period of 2 h at room temperature, and electrophoresis of 0.1 mL of the mixture at 2.5 mA/gel for about 2 h. Advantages include speed, quality of resolution, and low background. This method was compared with Amido Black post-electrophoresis staining in 35 sera. Of these, 16 showed identical results with respect to the number of bands. In the other 19, Amido Black showed more bands in the post-transferrin region and, sometimes, in the post-albumin region. The pre-stained gels showed slower electrophoretic mobilities of the components. Protein bands eluted from pre-stained gels retained immunological reactivity. (+info)