Coomassie Brilliant Blue

Coolwater Coomassie Brilliant Blue Cooperite Cooper pairs Coordination Coordination catalysts... 

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. 

To quantitate proteins from staining, a densitometer aided by computer software is used to evaluate band areas of samples compared to band areas of a standard curve. Amido black, Coomassie Brilliant Blue, and silver stains are all appHcable for use in quantification of proteins. 

Protein concentration can be determined using a method introduced by Bradford,4 which utilises Pierce reagent 23200 (Piece Chemical Company, Rockford, IL, USA) in combination with an acidic Coomassie Brilliant Blue G-250 solution to absorb at 595 nm when the reagent binds to the protein. A 20 mg/1 bovine serum albumin (Piece Chemical Company, Rockford, IL, USA) solution will be used to prepare a standard calibration curve for determination of protein concentration. The sample for analysis of SCP is initially homogenised or vibrated in a sonic system to break down the cell walls. 

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". 

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. 

Fig. 7. Coomassie Brilliant Blue R-250 (I) is used to stain proteins, e.g., after gel-electrophoretic separation, its derivative G-250 (II) is applied in the Bradford assay for protein quantification... 

Coomassie brilliant blue has been used extensively in a general quantitative method for proteins, and when complexed with protein shows a shift in... 

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

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

SDS-PAGE was performed essentially by the method of Laemmli and Favre (39) using 3% and 7.5% (w/v) total acrylamide concentrations in the stacking and resolving gels respectively. Sodium dodecyl sulphate was at a concentration of 0.1% (w/v). Staining for protein was accomplished using Coomassie Brilliant Blue R-250, while peroxidase activity was localized by the method of Thomas et al. (40). 

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. 

Staining Solution Dissolve 0.25g of Coomassie Brilliant blue R250 in 125 ml methanol, before adding 25 ml of acetic acid and 100 ml of water... 

Tests of Purity, Isoelectric focusing (lEF) in a 0.5-mm thick horizontal slab gel was performed with LKB pH 7-9 ampholyte (Ampholine 1809-136) (5). Electrophoresis was run at 10°C for 6 h at a constant voltage of 1800 V. Protein was visualized using silver stain (9) or Sigma Coomassie Brilliant Blue G-250. 

A 0.1 g Coomassie Brilliant Blue G 250 (C.1.42655 ) are dissolved in 50 ml 50% ethanol (v/v). After that, 100 ml of 85% phosphoric acid are added and made up with ddH20 to a total volume... 

Addition of a trace of Coomassie Brilliant Blue R250 facilitates the sample application and indicates the electrophoresis front For peptides < 5 kD... 

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

Marker proteins with known isoelectric point should he used to check the pH gradient. These proteins are stained hy the common protocols (e.g., Coomassie Brilliant Blue, Protocol 2.3.1.2, or silver staining. Protocol 2.3.2). 

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