1
Brilliant Blue G250
Coomassie Brilliant Blue
and Mechanisms of Protein Staining and
Bradford-Assay
ORIGIN
The name Coomassie was first used in the late 19th century, adopted from the town of Coomassie (modern-day
Kumasi in Ghana), as a trade name of the dye manufacturer Levinstein Ltd. for two similar triphenylmethane dyes
used as acid wool dyes. The two blue dyes were then first produced in 1913 by Max Weiler based in Elberfeld,
Germany. Today, the term ‘CoomassieTM
’ is a registered trademark of Imperial Chemical Industries.
Overall, there are approx. 40 dyes called ‘CoomassieTM
xy’, while only Coomassie
TM
G250 and CoomassieTM
R250
play a crucial role in biochemical analyses. During the last years, however, most authors referred to these dyes
simply as ‘Coomassie
TM
’ without specifying which dye is actually meant.
The term ‘250’ originally was used for denotation of the purity of the
dye. The suffix ‘G’ in ‘Brilliant Blue G250’ was added to describe the
slightly greenish colour of the blue dye. The suffix ‘R’ in ‘Brilliant Blue
R250’ is an abbreviation for ‘red’ as the blue colour of the dye has a slight reddish
tint. Coomassie
TM
Brilliant Blue G-250 differs from CoomassieTM
Brilliant Blue R-250
by the addition of two methyl groups.
BACKGROUND OF COLOUR CHANGES
The colour of the two dyes depends on the acidity of the solution and on its binding status to amino acids or
peptides. At a pH of less than 0 the dye has a redcolour with an absorption maximum at a wavelength of 470 nm.
At a pH of around 1 the dye is greenwith an
absorption maximum at 620 nm while above
pH 2 the dye is bright bluewith a maximum at
595 nm.
The different colours result from the differently
charged states of the dye molecule, corresponding to the amount of positive charges at the three nitrogen atoms
present, while the two sulfonic acid groups are normally always negatively charged.
• At a pH of around zero, all three nitrogen atoms are positively charged, thus the dye will be a cation with an
overall charge of +1, being in the redform.
• In the greenform (pH of approx. 1) the dye will have no net overall charge (+2 and -2).
• At pH of 2 and more, up to the neutral pH, only one nitrogen atom carries a positive charge and the dye molecule
is a blueanion with an overall charge of -1.
• Under alkaline conditions, the final proton is lost and the dye becomes pinkin colour. This state, however, is of
no relevance in biochemical assays.
MECHANISM OF GEL STAINING
Visualisation of proteins by Coomassie Brilliant Blue R-250 was first performed 1963 by Fazekas de St. Groth and
colleagues (Fazekas de et al.(1963) Biochim. Biophys. Acta71:377-91). Two years later, Meyer and Lambert used
Coomassie Brilliant Blue R-250 to stain proteins in a polyacrylamide gel (Meyer and Lambert (1965) Biochim.
Biophys. Acta107:144-5).
CoomassieTM
Brilliant Blue forms strong but non-covalent complexes with proteins, most probably based on a
combination of van der Waals forces and electrostatic interactions. Formation of the protein/dye complex stabilises
the negatively charged anionic form of the dye producing the blue colour which may then be seen on the membrane
or in the gel. The bound number of dye molecules is approx. proportional to the amount of protein present per band.
However, binding of the CoomassieTM
dyes to basic amino acids is much more efficient then to acidic amino acids;
this effect may cause slight differences in staining of proteins in gels. When standard staining is used the gel matrix
has to be destained subsequently, in order to visualize protein bands. Modern gel staining solutions use a colloid
form of the ‘G’ dye in solutions containing phosphoric acid (e.g. Roti
®
-Blue, Art. No. A152), in order to avoid the
necessity to destain the gel (Diezel et al.(1972) Anal. Biochem.48:617-20).
MECHANISM OF BRADFORD ASSAY
As mentioned above, the dye molecules bind to proteins to form a protein-dye complex. The Bradford assay uses
the spectral properties of Coomassie Brilliant Blue G-250 to estimate the amount of protein in a solution (Bradford
M.M. (1976) Anal. Biochem.72:248-54). In Bradford solutions, the dye is kept at a low pH in the red or greenish
form; due to that mixture the Bradfordsolution itself looks brown. Again, formation of the protein/dye complex
stabilises the negatively charged anionic form of the dye producing the blue colour. The optical absorbance of the
solution is measured at a wavelength of 595 nm. Thus, under
standard conditions (proteins in PBS or other low concentrated salt
solutions), highly linear standard curves may be produced.
This process, however, is altered by several putative factors. Binding
of the Coomassie
TM
dyes to basic amino acids is much more efficient
then to acidic amino acids. Thus, measurement of proteins with
identical concentrations but significantly different amounts of basic
amino acids may cause different data. Further, the binding to amino
acids itself as well as the stabilization of the cationic, acidic form is
hindered by several reagents that may be present in the assay like detergents, biological buffers, sugars etc. The
dye also forms a complex with the anionic detergent
sodium dodecylsulfate that stabilizes the neutral green
form of the dye. These processes result in a signal shift
during measurement that may well completely prohibit
reliable analysis of the data.
