Coomassie brilliant blue dye

Excitable tissue preparations were obtained fresh daily from live animals using the technique described by Dodd et al. (12). Protein was measured on each synapto-some preparation using the Coomassie Brilliant Blue dye technique described by Bradford (13) results were expressed as "toxin bound per mg synaptosome protein". 

Procedure 11.4 Quantitation of protein using Coomassie brilliant blue dye... 

Neuhoff, V., Stamm, R., Pardowitz, I., Arold, N., Ehrhardt, W., Taube, D. (1990). Essential problems in quantification of proteins following colloidal staining with coomassie brilliant blue dyes in polyacrylamide gels, and their solution. Electrophoresis 11,101-117. 

The Bradlord assay (named after its inventor] uses Coomassie brilliant blue dye. a histological slam which binds to protein and as the name surjgests. turns a brighi blue colour. At low concentrations Ihe absorbance is proportional to Ihe protein concentration in solution. 

Electrophoresis A family of separation methods based on the motion induced in particles by an applied, uniform electric field. The most common application of electrophoresis is gel electrophoresis, in which sample (typically proteins, amino acids, nucleic acids, etc.) is applied to a channel that is formed in a cross-linked polymer, usually polyacrylamide or agarose (the gel). The speed at which the individual species move through the gel under the influence of the field is determined largely by the size of the species, as expressed by the mass-to-charge ratio. After separation, the individual species usually appear as discrete bands that may be better visualized by staining with ethidium bromide, silver, or Coomassie Brilliant Blue dye. Other related... 

Amido black is a commonly used stain, but it is not very sensitive. It is often used to visualize concentrated proteins or components that are readily accessible to dyes such as proteins that have been transferred from a gel to nitrocellulose paper. Two of the more sensitive and more frequently used stains are Coomassie Brilliant Blue (R250 and G250) and silver stains. Because these stains interact differently with a variety of protein molecules, optimization of the fixative and staining solutions is necessary. The Coomassie stains are approximately five times more sensitive than amido black and are appropriate for both agarose and polyacrylamide gels. The silver stain is approximately 100 times more sensitive than Coomassie and is typically used for polyacrylamide gels. 

Coomassie Brilliant Blue G250 dye Binds specifically to tyrosine side chains of proteins Cell membrane No [33]... 

The most common methods used to determine protein concentration are the dye-binding procedure using Coomassie brilliant blue, and the bicinchonic-acid-based procedure. Various dyes are known to bind quantitatively to proteins, resulting in an alteration of the characteristic absorption spectrum of the dye. Coomassie brilliant blue G-250, for example, becomes protonated when dissolved in phosphoric acid, and has an absorbance maximum at 450 nm. Binding of the dye to a protein (via ionic interactions) results in a shift in the dye s absorbance spectrum, with a new major peak (at 595 nm) being observed. Quantification of proteins in this case can thus be undertaken by measuring absorbance at 595 nm. The method is sensitive, easy and rapid to undertake. Also, it exhibits little quantitative variation between different proteins. 

Direct staining of proteins (e. g., after electrophoretic separation in polyacrylamide gels) can be achieved by treatment with dyes like Coomassie Brilliant Blue R-250 [146] (Fig. 7), which binds positively charged proteins in an acidic fixation buffer, allowing detection down to 0.1 pg of protein. 

Figure 11.11 Absorption spectrum of the protein-Coomassie brilliant blue G 250 complex A, Coomassie brilliant blue only B, protein-dye complex.

Bradford reagent Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol and add 100ml of 85% (w/v) phosphoric acid. Dilute to 1 litre when the dye has completely dissolved, and filter through Whatman 1 paper just before use... 

The dye Coomassie Brilliant Blue R250 nonspecifically binds to all the protein. The gel is soaked in the dye for it to seep in and bind to the proteins. The gel is then destained to remove the unbound dye. The dye binds to the protein and not the gel, and hence the protein bands can be visually seen. The binding of the dye to the protein is approximately in stoichiometry, so the relative amounts of protein can be determined by densitometry. For most SDS and native gels, separated proteins can be simultaneously fixed and stained in the same solution. 

As tracking dye Coomassie Brilliant Blue G-250 is recommended. Bromophenol blue does not suit well since it moves significantly slower than small peptides. 

Gels are stained very sensitive and quick by combination of Coomassie Brilliant Blue R250 with Bismarck Brown R (Basic Brown4 C.1.21010). In our opinion, this is the best staining method using organic dyes. 

A variety of methods are available to detect proteins separated by electrophoresis or to measure the concentration of total protein in a solution. These methods are normally based on the binding of a dye to one of the amino acids in protein, or a color reaction with an amino acid side chain. The most commonly used stains for protein detection on gels are Coomassie Brilliant Blue (98) and silver stain (99,100). These methods detect any protein residues, either in solution or on an electrophoresis gel. Their main requirement is sensitivity, not specificity. New, more sensitive dyes are being developed for the proteomic analysis of protein structure and sequence, for example Ruby Red (101). 

A third widely used procedure, introduced by Bradfords and modified by others, measures the binding of the dye Coomassie brilliant blue whose peak absorption shifts from 465 nm to 595 nm upon binding. The change occurs within two minutes and is stable. However, the color yield varies from one protein to another. 

Detection of proteins on thin-layer plates, gel slabs, or membranes is often accomplished by staining with a dye,265-267 the most widely used being Coomassie brilliant blue.268 Various silver-containing stains may also be used. After separation of a protein mixture by electrophoresis and transfer to an inert membrane,... 

Bovine gamma globulin standard, 0.1 mg/mL in H20 Bradford dye reagent. This is a commercially available mixture of Coomassie Brilliant Blue G-250 dye, phosphoric acid, and methanol. Spectrophotometer for reading A595 with glass cuvettes (1 or 3 mL)... 

Coomassie Brilliant Blue G-250 dye, phosphoric acid, and methanol. 

Coomassie brilliant blue staining (see Support Protocol 1) is based on the binding of the dye Coomassie brilliant blue R250, which... 

The dye l-Anilino 8-Naphthalene Sulfonic acid (ANS) has high specificity for protein. It fluoresces only when bound to protein [30]. In smears and handsections (i.e. unembedded materials) we have never observed it to effect emulsion stability in the manner more traditional protein dyes such as Coomassie Brilliant Blue or Fast Green often do. This relative pH independence probably is due to the mode of action of this dye. It becomes fluorescent in hydrophobic pockets on protein molecules [30] in contrast to the ionic bonding necessary for Fast Green FCF and Coommassie Blue [22]. We have not observed a strong cross-reaction with lipids, either, although a fluorescence of different spectral characteristics sometimes is seen. 

For sections of embedded material, we generally do not use ANS to localize protein. Instead we use Coomassie Brilliant Blue or Fast green FCF which are used as diachromes. Either dye is used at 0.1%W/V in 7% acetic acid [22], Slides having a puddle of stain over the sections are gently warmed for up to 5 minutes, rinsed with distilled water and allowed to dry before mounting in immersion oil for observation. Figure 5 shows an example of cream cheese treated in this manner to reveal protein. 

The most frequently used protein assay is based on a method after Bradford (Bradford, 1976), which combines a fast and easily performed procedure with reliable results. However, the Bradford assay has sensitivity limitations and its accuracy depends on comparison of the protein to be analyzed with a standard curve using a protein of known concentration, commonly bovine serum albumin (BSA). Many commercially available protein assays such as those from Pierce or BioRad rely on the Bradford method. The assay is based on the immediate absorbance shift from 465 nm (brownish-green) to 595 nm (blue) that occurs when the dye Coomassie Brilliant Blue G-250 binds to proteins in an acidic solution. Coomassie dye-based assays are known for their non-linear response over a wide range of protein concentrations, requiring comparison with a standard. The dye is assumed to bind to protein via an electrostatic attraction of the dye s sulfonic groups, principally to arginine, histidine, and lysine residues. It also binds weakly to the aromatic amino acids, tyrosine, tryptophan, and phenylalanine via van der Waals forces and hydrophobic interactions. 

Dye-Binding (Bradford) Assay. The binding of proteins to Coomassie Brilliant Blue 250 causes a shift in the absorbance maximum of the dye from 465 nm to an intense band at 595 nm. Determination of the increase in absorbance at 595 nm as a function of protein added provides a sensitive assay... 

Detection and analysis Once the electrophoretic separation is completed, the gel is treated with a stain, most popularly Coomassie Brilliant Blue, to reveal protein bands. In the case of Coomassie Brilliant Blue, this process involves a stain-destain cycle, and as proteins can differ in their affinity to take up this dye the detection is really only considered qualitative. There are various other alternative stains which include Amido black. Ponceau red and silver nitrate. Of these, silver staining is widely considered to be one of the most sensitive staining... 

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