Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Abstract

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.

The Radical S-Adenosyl-l-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds

“…The reaction mixture (2 ml) was applied to a His trap column (2 ml) (QhpD contained N-terminal H 6 -tag), and the flow-through (FT, 2 ml), first and second wash (W1 and W2, each 2 ml), and eluted (E, 2 ml) fractions were collected and analyzed by SDS-PAGE (10 l/lane). The protein bands were stained with colloidal Coomassie G-250 (54). B, enhanced image of the boxed area in A.…”

Physiological and Proteomic Responses of Continuous Cultures of Microcystis aeruginosa PCC 7806 to Changes in Iron Bioavailability and Growth Rate

“…Protein pellets were air-dried and resuspended in dissolving buffer (50 mM NaHCO 3 , 0.08% SDS, MilliQ water) for quantification via the bicinchoninic acid (BCA) assay. A portion (15 g) of each sample was separated by SDS-PAGE (NuPAGE 4% to 12% Bis-Tris gradient gels; Invitrogen) and visualized by colloidal Coomassie staining (35).…”

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

“…FLAG-tagged ORF24 was purified from whole-cell extract using FLAG resin (Sigma), washed five times in lysis buffer, and eluted from the resin with 1ϫ FLAG peptide (Elim BioPharm). Protein purity was assessed by colloidal Coomassie staining (18). Purified protein was dialyzed against 20 mM HEPES (pH 7.9), 40% glycerol, 100 mM KCl, 0.5 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF).…”