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Application Note #2 Bio-Imaging Systems Coomassie Brilliant Blue Introduction Protein stains are based on the differential binding of the stain by the protein and the matrix. The main characteristics for an efficient stain are high sensitivity, low background, large linear range, and ease of use. The sensitivity of a given dye depends on the extension coefficient and the avidity, which determines the linear range of detection for that protein. The Coomassie Brilliant Blue dye which is commonly used in SDS-PAGE, was first described by the German scientist Volker Neuhoff. The dye gets its name from the African city Kumasi, formerly Coomassie, a city in central Ghana. Currently, there are two kinds of the Coomassie dyes: R-250 and G-250. Coomassie R-250 is the more commonly used and sensitive of the two. Though less sensitive, Coomassie G-250 has particular properties that enable it to be used to create a rapid and convenient staining procedure. In the staining reaction, the Coomassie dye binds to proteins through ionic interactions between sulfonic acid groups and positive protein amine groups through Van der Waals attractions. DNR developed unique Bio-Imaging systems that can easily detect the Coomassie Brilliant Blue dye in a quick, accurate and reproducible manner. Our superior GelCapture software and camera, allow the user to detect both the highest and lowest band intensity. Coomassie Brilliant Blue Forms Coomassie Brilliant Blue-R Coomassie Brilliant Blue-G Image courtesy Sigma-Aldrich Co. Detection There is a shift in the peak absorbance of the dye when it binds to a protein. Unbound Coomassie Blue absorbs light maximally at a wavelength of 465 nm, while the absorption maximum is at 595 nm when the dye is bound to protein. Coomassie Brilliant Blue-Protein Complex Absorbance Spectrum 2.0 Absorbance 1.8 1.2 DNR Laboratories Detection Tip The Coomassie Brilliant Blue dye is best detected by using the 470nm excitation LED, and UV High filter for emission (Instead of the traditional lower white light). There are several configurations which can be used to detect this dye, and the user must choose the ultimate parameters for his experiments, according to his experiment specification and the Bio-Imaging System. The general parameters for Coomassie Brilliant Blue dye detection in the F-ChemiBIS 6 Pro Bio-Imaging System are as follows: Exposure Time Ms: 250 0.8 Binning 1 0.4 Gain 3 Filter High UV Illumination 470nm LED + Trans 312nm UV Drawer UV + converter 0 400 500 600 700 800 Wave length (nm) Image courtesy the Research Ser vices Branch Application Note #2 | Coomassie Brilliant Blue This DNR Coomassie Brilliant Blue image was captured using the F-ChemiBIS 6 Pro Bio-Imaging System with a Gradient SDS PAGE 4-15% gel and a Coomassie Brilliant Blue-R, Sigma-Aldrich catalog number B8647. The sample was an extraction of Human MEK1 protein from E. Coli cellular lisate. Bibliog raphy Blakesley, R.W.; Boezi , J.A . "A new Staining Technique for Proteins in Polyacr ylamide Gels Using Coomassie Brilliant Blue G250," Anal Biochem 1977, 82 (2), 580-582. Syrovy, L.; Hodny, Z. "Staining and Quantification of Proteins Separated by Polyacr ylamide Gel Electrophoresis,". J. Chromatog. 1991, 569, 175196. Tal,M.; Silberstein , A.; Nusser , E. "Why Does Coomassie Brilliant Blue R Interact differently with different proteins?," The Journal of Biological Chemistr y 1985, 260 (18), 9976-9980. Volker N.; Reinhard S.; Hansjörg E. "Clear Background and Highly Sensitive Protein Staining with Coomassie Blue Dyes in Polyacr ylamide Gels: A Systematic Analysis," Electrophoresis . 1985, 6(9), 427-448. Bio-Imaging Systems Tel: +972 2 570-0818 U.S. toll-free: 1 866 300-4286 [email protected] This Technical Note is provided as a guideline, and is not intended to be a guarantee of performance. www.dnr-is.com Advanced Technologies for Breakthrough Results