Application Note #2 Bio-Imaging Systems Coomassie Brilliant Blue

Introduction

Protein stains are based on the differential binding of commonly used and sensitive of the two. Though less the stain by the protein and the matrix. The main sensitive, Coomassie G-250 has particular properties characteristics for an efficient stain are high sensitivity, that enable it to be used to create a rapid and low background, large linear range, and ease of use. convenient staining procedure. In the staining reaction, The sensitivity of a given dye depends on the extension the Coomassie dye binds to proteins through ionic coefficient and the avidity, which determines the linear interactions between sulfonic acid groups and positive range of detection for that protein. protein amine groups through Van der Waals The Coomassie Brilliant Blue dye which is commonly attractions. used in SDS-PAGE, was first described by the German scientist Volker Neuhoff. The dye gets its name from DNR developed unique Bio-Imaging systems that can the African city Kumasi, formerly Coomassie, a city in easily detect the Coomassie Brilliant Blue dye in a central Ghana. quick, accurate and reproducible manner. Our superior Currently, there are two kinds of the Coomassie dyes: GelCapture software and camera, allow the user to R-250 and G-250. Coomassie R-250 is the more 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 DNR Laboratories Detection Tip when it binds to a protein. Unbound Coomassie Blue absorbs light maximally at a wavelength of 465 nm, The Coomassie Brilliant Blue dye is best detected by while the absorption maximum is at 595 nm when the using the 470nm excitation LED, and UV High filter dye is bound to protein. for emission (Instead of the traditional lower white light). There are several configurations which can be Coomassie Brilliant Blue-Protein used to detect this dye, and the user must choose the Complex Absorbance Spectrum ultimate parameters for his experiments, according to his experiment specification and the Bio-Imaging System. The general parameters for Coomassie Brilliant Blue 2.0 dye detection in the F-ChemiBIS 6 Pro Bio-Imaging

1.8 System are as follows:

1.2 Exposure Time Ms: 250

0.8 Binning 1 Absorbance 0.4 Gain 3

0 Filter High UV 400 500 600 700 800 Wave length (nm) Illumination 470nm LED + Trans 312nm UV Drawer UV + converter Image courtesy the Research Services 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.

Bibliography Blakesley, R.W.; Boezi , J.A . "A new Staining Technique for Proteins in Polyacrylamide Gels Using Coomassie Brilliant Blue G250," Anal Biochem 1977, 82 (2), 580-582. Syrovy, L.; Hodny, Z. "Staining and Quantification of Proteins Separated by Polyacrylamide Gel Electrophoresis,". J. Chromatog. 1991, 569, 175- 196. Tal,M.; Silberstein , A.; Nusser , E. "Why Does Coomassie Brilliant Blue R Interact differently with different proteins?," The Journal of Biological Chemistry 1985, 260 (18), 9976-9980. Volker N.; Reinhard S.; Hansjörg E. "Clear Background and Highly Sensitive Protein Staining with Coomassie Blue Dyes in Polyacrylamide Gels: A Systematic Analysis," Electrophoresis. 1985, 6(9), 427-448.

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