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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
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This Technical Note is provided as a guideline, and is not intended to be
a guarantee of performance.
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