Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.Several improvements of the original Coomassie protocol 1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution 2 , and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol 3 . Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol 4 . The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

Proteins separated by two-dimensional gel electrophoresis can be visualized by in-gel detection using -different staining methods. Ideally, the dye should bind non-covalently to the protein following a linear response curve. Since protein concentrations in biological systems may vary by six or more orders of magnitude (Corthals GL et al., Electrophoresis 21(6):1104-1115, 2000), the staining should allow for a detection of very low protein amounts. At the same time, saturation effects have to be avoided because they impede normalized quantification.Most proteomics laboratories apply Coomassie, silver, or fluorescent stains. Using the colloidal properties of Coomassie dyes, detection limits at the lower nanogram level can meanwhile be achieved. Characteristics like ease of use, low cost, and compatibility with downstream characterization methods such as mass spectrometry, therefore, make colloidal Coomassie staining well suited for the in-gel detection method in quantitative proteomics.

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