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Coomassie G is more sensitive Coomassie R provides better resolution and lower cost. Introduction to Polyacrylamide Gels. Imaging and Analysis of 2-D Electrophoresis Gels.
Image Lab Software Suite. Though less sensitive, Coomassie G can be used in place of the R form to create a rapid and convenient staining procedure. This capability of G is due to its particular properties. Coomassie G manifests a leuco form below pH 2. Solutions of the dye, dark blue black at pH 7, turn a clear tan upon acidification.
The leuco form recovers its blue color upon binding to protein, apparently due to the more neutral pH of the environment around the protein molecule. Under proper conditions, a gel placed in an acidified solution of Coomassie G will manifest blue protein bands on a light amber background. The bands develop rapidly and there is no need to destain, for the background color is so light as to be essentially clear. This stain is less sensitive than Coomassie R protocols, detecting 0.
It is an organic dye that makes complexes with basic amino acids, such as lysine, histidine, tyrosine, and arginine. The stain transfers an overall negative charge to the proteins allowing their separation from the polyacrylamide gel. Different kinds of Coomassie dyes are available. In its anionic form, the CBB dye forms a stable blue complex with the proteins. The CBB staining is capable of detecting as little as ng of protein, but the sensitivity of the method could be improved by performing it at elevated temperatures.
The test could also be used with a variety of proteins and polypeptides having molecular weights greater than The assay is rapid and offers high reproducibility, excellent sensitivity, and ease of use. The Coomassie Brilliant Blue G dye has three forms: anionic blue , neutral green , and cationic red.
In an acidic environment, the red dye is converted into its blue form after binding to the protein of interest. If no protein binds to the dye, then the solution will remain brown. The CBB stain forms a strong, noncovalent complex with the carboxyl group of the protein by van der Waals force and the amino group through electrostatic interactions.
These pockets non-covalently bind to the non-polar components of the dye via van der Waals forces. The ionic interaction further strengthens the bond. This complex formation enables the detection of the proteins separated by the gel. Quantification of proteins Ku. The Coomassie brilliant blue stain is used in the Bradford assay, a colorimetric protein assay, to quantify the proteins separated by gel.
This assay is performed by determining the absorbance shift of the Coomassie Brilliant Blue G The amount of the complex present in the solution is used as an indicator for the protein concentration by measuring the intensity of the blue color after stabilization.
In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue CBB staining remains the most commonly used detection technique for proteins separated by electrophoresis [5] , [7]. CBB staining was first developed to stain proteins on a cellulose acetate sheet in [8].
Coomassie blue staining has the following advantages: low cost, visual inspection, easy operation, convenient scanning procedure for image acquisition, better suitability for quantitative analysis than silver staining, and the capacity to allow possible modifications for fast or highly sensitive staining. However, it still requires a long staining time, and the relatively complicated ingredients make its use inconvenient. In the present study, we present an improved method for in-gel staining of proteins, which has the advantage of speed over the conventional CBB staining, yet has a comparable sensitivity.
Acrylamide and bis-acrylamide were purchased from Sigma St. All reagents used were of analytical grade. CBB R mg was dissolved in ml of distilled water by stirring for 2—4 h. The stacking and separating gels used were 3. The running buffer was prepared from 25 mM Tris base, 0. Concentrations of BSA were determined by the Bradford method [15]. Prior to electrophoresis, the samples were heated in the presence of sample buffer 70 mM Tris—HCl, pH 6. Subsequently, the stained gels were removed from the staining solution and boiled in distilled water for 30—60 s.
The washing step was repeated several times. The Neuhoff's Chinacolloidal Coomassie Blue G staining has a detection limit of approximately 10 ng of protein per spot [16]. The results showed that this approach has a sensitivity of 10 ng, which is the same as the traditional Coomassie blue stains [16] and other fast Coomassie stains [15] , [17] Fig.
To evaluate whether the acids have any effects on the protocol, we used different acids, including hydrochloric acid, phosphoric acid, and acetic acid, to prepare the staining solution in the experiment. No significant difference was found between staining with different acids solutions and with no acid solution. The water solution with and without acid could dye the protein just as well in this staining protocol Fig. All the traditional staining solutions contain methanol, acetic acid, or phosphoric acid which not only produce unpleasant smell but also cause environmental pollution.
In the dye stain, no acids are needed for the staining solution preparation. Added to the water-soluble CBBR staining solution were 0. Temperature is a very important factor for protein detection in the protocol. We compared the gel staining under different boiling times and different staining times at room temperature. Boiling for 30—60 s was found to be enough to obtain a clear band Fig.
This indicates better time-saving ability compared with conventional Coomassie [16] and other fast Coomassie staining methods [15] , [17]. However, staining with this quick staining solution at room temperature was difficult, and no clear bands were acquired even after staining for 24 h Fig. The color of the bands and backgrounds became more unclear as the staining time increased. The backgrounds were very difficult to retain Fig. Therefore, boiling temperature was necessary to obtain a clear band and clean background.
Additionally, van der Waals forces and hydrogen bonding contribute to the binding interaction between dye and protein [18]. The mechanism for hot fast CBB staining with the rise of temperature is possibly because of the increasing speed of the molecules of the dye and proteins.
Washing before staining is necessary to prepare a clean background.
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