Coomassie+Blue+Staining

Shanice Brown (Coomassie Blue Staining)
 * What is Coomassie Blue Staining?**

Coomassie Blue is a stain used in protein gel analysis. The stain binds to proteins through ionic interactions of sulfonic acid groups in the dye and positive amine groups in the protein. The stain allows the gel to express a banding pattern. Coomassie Blue, or Brilliant Blue, is a stain that works at a specific pH. The pH in which we see the stain as a "brilliant blue" is a pH of 2 or higher. Because the dye can form strong complexes with the protein within a gel, it allows for stabilization of anionic charge of the blue dye. The dye becomes visible in the membrane of the gel in which it is used. The number of dye ligand molecules that are depicted in the dye, bind to proteins approximately proportional to the amount of protein present in a band ("Coomassie Brillant Blue R-250 and G-250 dyes", 2014). The dye later turns to a reddish brown color then a brillant blue due to protein binding with an absorption value of 585 nm ("Coomassie Brillant Blue R-250 and G-250 dyes", 2014).

The Coomassie dyes exist in two forms (R-250 and G-250) that bind to proteins through Van Waals attractions between ionic interactions of sulfonic group dye and positive amine groups ("Staining Protein Gels with Coomassie Blue", 2011). Shown in Figure 1 and 2. The R-250 Coomassie blue dye can be use to detect proteins as small as 0.1 micrograms.The R-250 Coomassie blue dye has a red tinted shade and G-250 Coomassie Blue dye has a green tinted shade. The R-250 Coomassie Blue Dye lacks two methyl groups that are present in G-250 Coomassie Blue dye. Both the R-250 and G-250 Coomassie Blue dyes bind to specific proteins through basic amino acids (arginine, lysine, and histidine) ("Coomassie Brillant Blue R-250 and G-250 dyes", 2014). Both R-250 and G-250 can be dissolved in methanol but G-250 can also be dissolved in ethanol. R-250 Coomassie dye is insoluble in water and G-250 is slightly soluble in water ("Coomassie Brillant Blue R-250 and G-250 dyes", 2014).

Figure 1- Coomassie Blue R-250

Figure 2- Coomassie Blue G-250



http://www.piercenet.com/media/GelCodeBlueGels.jpg

http://www.invitrogen.com/etc/medialib/en/images/ics_organized/applications/proteins_expression/data_image/560_wide.Par.32783.Image.-1.0.1.gif

Protocol of Making a Protein Stain (Coomassie R-250 Brilliant Blue)

1. Obtain 45% Methanol (reagent grade), 10% Glacial Acetic Acid, 45% water, and 3g/L Coomassie R-250 Brillant Blue 2. Add 100 mL of 10% Glacial Acetic Acid to 450 mL pure 45% water 3. Dissolve about 3 grams of Coomassie Dye in 450mL methanol. 4. Filter the solution before using the solution

References ====Coomassie Brilliant Blue R-250 and G-250 Dyes (2014). In //Thermo Scientific Pierce Protein Biology //. ==== ==== from http://www.piercenet.com/product/coomassie-brilliant-blue-r-250-g-250 ====


 * Protocol: (**http://www.mass-spec.siu.edu/CoomassieStainingProtocol.pdf**)**

Protocol for Staining Gels with Coomassie Blue G-250

1. Remove the gel from the electrophoresis chamber and place enough of 0.5% Coomassie Blue G-250 (prepared in 50% methanol/ 10% acetic acid) to cover the gel. Use freshly washed labware that has never been in contact with nonfat milk, BSA or any other protein blocking agent to prevent carryover contamination. Stain for about 5 minutes.

2. Discard stain and rinse briefly with MilliQ water to remove most of the residual stain in the glassware.

3. Destain with 40% HPLC grade methanol/ 10% acetic acid, replacing the solution every 10-20 minutes until faint bands are observed. Kimwipes rolled up into balls can be added to speed up the destaining.

4. Continue destaining with MilliQ water until bands are very clean. Usually we destain overnight in MilliQ water with several Kimwipes present. Bands/spots can now be excised and submitted for analysis.


 * Who and Where?**

Coomassie Blue dyes were first produced in 1913. The dyes are similar to a wool dye used by Levinstein Ltd. The man who produced these dyes is, Max Weiler. The name Coomassie comes from a region in Ghana by the same name (Kumasi). Today there are more than 40 Coomassie dyes and all are trademarked at Imperial Chemical Industries. These dyes are very important to biochemical research and are still used today by many scientists.

Current Research
 Coomassie Blue Staining technique is used in many biology research studies. In a research experiment, coomassie blue staining of proteins were compared to SYPRO Ruby (SR) staining with BioSafe and Neuhoff (NCCB) in one and two dimensional electrophoresis. In addition, the sensitivity of protein proteomes were measured. The second dimensional gel electrophoresis was used to analyze mouse and arabidopsis thaliana proteomes (Gauci et al, 2013). The experimental data yield that the Coomassie blue staining showed a lower abundance of protein in comparison to SR. Both coomassie blue staining and SR did not depict a significant level of sensitivity after the proteins were optimized overnight (Gauci et al, 2013). The mouse and arabidopsis thaliana proteomes were detect using the NCCB (Gauci et al, 2013). When Coomassie Blue was used with NCCB the protocol resulted in a more sensitive protein detection. The experiment research determined that the Coomassie Blue stain can depict sensitivity of the proteins that were optimized overnight (Gauci et al, 2013).  In another experiment, coomassie blue G-250 was purified using a counter current chromatography multiple dual mode which allowed impurities to be removed (Mekaoui et al, 2012). An image of coomassie blue G-250 is shown in Figure 2. The research experiment measured 200 mg of purified coomassie blue extracted from 1 g of crude coomassie blue using 150 mL of butanol, 70 mL of heptane, and 200 mL of water (Mekaoui et al, 2012). The results of the experiment indicated that the counter current chromatography could not purify the coomassie blue dye because the coomassie blue dye sample was continuously trapped in the counter current chromatography column. In addition, the researcher suggested that the solvent used in the experiment increased the levels of separation in the coomassie blue G-250 sample (Mekaoui et al, 2012).  In another research study, coomassie blue dye was used to detect polyomavirus virion found in infected cells of mouse such as VP1 protein and cellular complexes (Hornikova et al, 2011). The mouse polyomavirus cells were separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The results determined that bands were not present in the blue coomassie gel. The researchers stated that the BN-PAGE was beneficial tool to use to analyze different types of complexes formed by proteins. In addition, the method of using blue coomassie dye to detect new cellular proteins that are involved in virion assembly was not suitable (Hornikova et al, 2011). The analysis determined that the protein samples were made from viral proteins. The BN-PAGE was beneficial to detect the VP1 protein found in the nuclear lysates of the infected cells. The BN-PAGE detected several forms of VP1 proteins in the infected mouse cells (Hornikova et al, 2011). The coomassie blue stain dye was not proven to effective in this experiment. The coomassie blue stain dye was effectively used in Gauci et al experiment and Mekaoui et al experiment. The coomassie blue stain dye can be used to measure the proteins and protein sensitivity.

References:

Coomassie Brilliant Blue and Mechanisms of Protein Staining and Bradford-Assay (n.d.). In Technical Info Carol Roth.  from http://www.carlroth.com/website/de-ch/pdf/Coomassie_E.pdf Coomassie Brilliant Blue R-250 and G-250 Dyes (2014). In Thermo Scientific Pierce Protein Biology.  from [] Gauci, V. J., Padula, M. P., & Coorssen, J. R. (2013, September). Coomassie blue staining  for high sensitivity gel-based proteomics. Journal of Proteomics, 90(2), 96-106. Horníková, L., Man, P., & Forstová, J. (2011, December). Blue native protein electrophoresis  for studies of mouse polyomavirus morphogenesis and interactions between the major capsid protein VP1 and cellular proteins. Journal of Virological Methods, 178(1-2), 229-234. Mekaoui, N., Chamieh, J., Dugas, V., Demesmay, C., & Berthod, A. (2012, April 6). <span style="font-family: 'Times New Roman',serif; font-size: 12pt;"> Purification of Coomassie Brilliant Blue G-250 by multiple dual mode countercurrent chromatography. Journal of Chromatography A, 1232, 134-141. <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Staining Protein Gels with Coomassie Blue (2011). In National DiagonisticsElecctrophoresis. <span style="font-family: 'Times New Roman',serif; font-size: 12pt;"> from []coomassie-blue