title: Coomassie blue staining for high sensitivity gel-based proteomics.
abstract: Gel electrophoresis, particularly one- (1DE) and two-dimensional electrophoresis (2DE), remain among the most widely used top-down methods for resolving and analysing proteomes. Detection of the resulting protein maps relies on staining (i.e. colloidal coomassie blue (CCB) or SYPRO Ruby (SR), in addition to many others). Fluorescent in-gel protein stains are generally preferred for higher sensitivity, reduced background, and wider dynamic range. Although traditionally used for densitometry, CBB has fluorescent properties. Indeed, infrared detection of CCB stained protein was comparable to SR, with BioSafe (Bio-Rad) and the Neuhoff formulation (NCCB) identified as potentially superior to SR; a minor sensitivity issue encountered in gel-resolved proteomes; might have been due to the unified staining protocol used. Here the staining protocol for both CCB formulations was optimised, yielding improved selectivity without affecting sensitivity; the resulting linear dynamic range was similar for BioSafe and NCCB and somewhat better than SR. 2D gel-based analyses of mouse brain and Arabidopsis thaliana (leaf) proteomes indicated markedly superior spot detection using the NCCB formulation. Thus more sensitive, quantitative in-gel protein analyses can be achieved using NCCB, at a fraction of the cost.
authors: Molecular Physiology, and the Molecular Medicine Research Group, School of Medicine, University of Western Sydney, Sydney, Australia.
authors: Victoria J
authors: VJ
authors: Gauci
authors: Matthew P
authors: MP
authors: Padula
authors: Jens R
authors: JR
authors: Coorssen
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000737:chemistry* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry / Q000502:physiology; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry*; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012394:Rosaniline Dyes / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D029681:Arabidopsis Proteins; D009419:Nerve Tissue Proteins; D009942:Organometallic Compounds; D012394:Rosaniline Dyes; C515601:Sypro Ruby; C048139:Coomassie blue
keywords: 1DE; 2DE; AS; BGAL; CA; Coomassie; Fluorescence; Gel electrophoresis; IPV; IRFD; LDR; LLD; LYS; MS; NCCB; Neuhoff colloidal coomassie blue; PHOSB; Protein staining; S(2)/BG; SR; STI; SYPRO Ruby; TYRP; Top-down proteomics; ammonium sulphate; beta-galactosidase; carbonic anhydrase; infrared fluorescent detection; inter-protein variability; linear dynamic range; lowest limit of detection; lysozyme; mass spectrometry; one dimensional gel electrophoresis; phosphorylase b; signal squared to background ratio; soybean trypsin inhibitor; trypsinogen; two dimensional gel electrophoresis