Coomassie R-250 and G-250 dyes are two most typical chemical types of Coomassie dyes, as disulfonated
triphenylmethane compounds. The R-250 (red-tinted) lacks two methyl teams which might be current within the G-250 (greentinted) type. The “250” initially denoted the purity of the dye.
The totally different colors are a results of the totally different charged states of the dye molecule.
At a pH of lower than Zero the G250 dye caries all three nitrogen atoms with a optimistic cost, it has a purple color with an
absorption most at a wavelength of 465 nm. At a pH of round 1 the dye bears an general cost of +1 (the two
sulfonic acid teams are negatively cost, due to theirextremely low pKa), so the dye is inexperienced with an absorption
most at 620 nm whereas above pH 2 the dye is brilliant blue with a most at 595 nm. At pH 7 the nitrogen atom of the diphenylamine moiety carries a optimistic cost, with an general cost of −1, and the dye has an extinction
coefficient of 43 000 M−1 cm−1
.
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The dye interacts electrostatically with the amino and carboxyl teams of proteins (noncovalently), so from primarily fundamental
aminoacids (arginine principally, lysine, histidine). Hydrophobic interactions are additionally considerably concerned. (phenylalanine,
tyrosine, tryptophane).
When dissolved in 0.01M citrate buffer at pH 3.0, Coomassie has an absorption most at 555nm; protein-dye
complicated is characterised by a peak barely broader than that of the free dye with a most at 549nm. Therefore, certain
and unbound dye will be distinguished (precept of Bradford assay).
Sometimes Coomassie gel stains and protein Bradford-type assay reagents are formulated as very acidic options in 25 to
50% methanol. In acidic situations, the dye binds to proteins primarily by fundamental amino acids (primarily arginine, lysine and histidine), whereas the formation of the complicated stabilizes the negatively charged anionic type of the dye producing the blue color. The variety of Coomassie dye ligands certain to every protein molecule is roughly proportional to the variety of optimistic fees discovered on the protein. Protein-binding causes the dye to alter from reddish-brown to brilliant blue (absorption most equals 595nm).
The aqueous-solubility of the G-250 dye is taken to good account in protocols of colloidal Coomassie staining.
There’s an interference from SDS detergent, particularly with G250 dye (see various BC protein Assay for assaying
proteins with SDS).
coomassie staining resolution
Coomassie Sensible Blue staining resolution
Dissolve 1 g of Coomassie Sensible Blue (Bio-Rad) in 1 liter of the next resolution:
Methanol (50% [v/v])
Glacial acetic acid (10% [v/v])
H2O (40%)
Stir the answer for 3-Four hours after which filter by Whatman filter paper. Retailer at room temperature.
Reagent
Amount (for 100 mL)
Ultimate focus
Coomassie Sensible Blue R-250
 0.05 g
  0.05%
Methanol
 50 mL
50% (v/v)
Glacial acetic acid
 10 mL
10% (v/v)
H2O
to 100 mL
Dissolve the Coomassie Sensible Blue R-250 dye, after which filter by a Whatman No. 1 filter to take away any particulate matter. May be saved at room temperature.
Description: DRAQ5™ is the trade name of 1,5-bis{[2-(di-methylamino) ethyl]amino}-4, 8-dihydroxyanthracene-9,10-dione (DRAQ-5™ is the trade mark of Biostatus Ltd).
ReadiUseâ„¢ DRAQ5 Staining Solution *5 mM in Water*
