Coomassie blue is our most quantitative stain as shown in the adjacent plot of spot stain density (volume above background) versus total protein loaded (rat liver cytosol) for polypeptide spot 8 of MW 76,100, pI 8.1 in the validation. See Method Validation (PDF) for detailed results confirming the linearity and reproducibility of Coomassie blue staining of our large format 2D gels. For Coomassie, one μg of a purified protein gives a dark spot while 0.1 μg gives a faint but clearly visible spot. Coomassie blue is compatible with mass spectrometry. See Burgess Cassler, A. et. al. Clinical Chem. 35 : 2297 1989 (PDF) for validation of our standard format gels.
The example below is from an Escherichia coli sample prepared in urea sample buffer (left) and SDS boiling buffer (right) run identically on a pH 4-8 IEF gradient and 10% acrylamide slab gels. The advantage of carrier ampholine 2D electrophoresis (aka 2D SDS PAGE) is compatibility with SDS buffer and ease of sample preparation. Molecular weight standards (220, 94, 60, 43, 29, and 14 kDa) appear as bands on the right side of the gel labeled with press-on numbers. One isoelectric point marker (tropomyosin), added to each sample as an internal standard, is marked with a black arrow.
Two gel sizes are offered for 2DE.
1) Standard format (2D-ES-1) 2D electrophoresis is 13 cm long and 15 cm wide and is perfect for immunoprecipitations, subcellular fractionations, most bacterial preparations and many proteomic applications. The maximum load is 200 μg protein in a maximum volume of 50 μl.
2) LARGE FORMAT 2D ELECTROPHORESIS (2D-ES-5) is 20 cm wide by 22 cm long. The larger size gives better resolution for proteomics applications; heavier loads may be applied to obtain more material for mass spectrometry. The maximum load is 600 μg protein in a maximum volume of 150 μl. One Coomassie blue-stained 2D gel with dimensions (dried) of 20 cm wide by 22 cm long is returned for each sample along with a pH gradient plot and a method description sheet.
Our standard IEF tube gel contains 2% pH 3-10 Isodalt Servalytes. Molecular weight standards (220, 94, 60, 43, 29, and 14 kDa) appear as bands on the right side of the gel labeled with press-on numbers. One IEF marker (tropomyosin), added to each sample as an internal standard, is marked with a black arrow. Gels are transparency dried unless otherwise requested. Custom pH gradients are available. Typical protein loads are 500 μg in 25-150 μl.
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