National Biochemicals, LLC. 1780 Enterprise Parkway • P.O. Box 1986, Twinsburg, OH 44087, USA Phone: 330. 425. 2522 • Fax: 330. 425. 2583 www.nationalbiochem.com COOMASSIE BRILLIANT BLUE G-250 Product No.: MB1131 Synonym: Coomassie™ G2501,2, CAS No.: 6104-58-1 Coomassie™ Brilliant Blue G250, CBBG, Storage Temperature: Ambient Serva Blue G™, Acid Blue 90

O The latter forms a stable complex with basic amino acid + S O- residues of proteins, mainly with arginine and aromatic H C N 3 O amino acids. Although the anionic form exists in negligible concentration at the pH of the assay reaction mixture, the binding to protein shifts the equilibrium toward its formation. CH 3 CH H3C 3 O Bradford reagent is prepared with Brilliant Blue G and is O supplied with usage instructions. It has been reported that the S ONa presence of vanadate (orthovanadate ion) in concentrations N N above 20 to 40 μmol/l in protein samples interferes with the H O Bradford microassay, but not the Bradford standard (non- H C 4 2 3 micro assay) method. The presence of detergents such as SDS and strongly alkaline buffering agents also interfere with this protein assay. PRODUCT SPECIFICATIONS Brilliant Blue G has been used for determining critical Molecular Formula: C H N Na S 5 47 48 3 7 2 micelle concentration of detergents. Molecular Weight: 854.0 Appearance: Deep Blue, Crystalline Powder PREPARATION INSTRUCTIONS Solubility (1% in Water): Complete, Deep Blue Solution Lambda Max: About 586 nm This product is soluble in water (1 mg/ml), yielding a dark Dye Content (Min.): 80% blue solution. Heat may be required to completely dissolve the material. PRODUCT DESCRIPTION STORAGE & STABLITLY The “G” in the name Brilliant Blue G refers to a blue dye with a greenish hue. The “R” in the name of brilliant blue R Store at room temperature. refers to a blue dye with a reddish hue.

Brilliant Blue G has been used in the Bradford dye-binding PROCEDURE protein assay.3,4 A mechanism for dye binding to protein has been proposed,4 based on measuring Coomassie Brilliant For staining proteins in polyacrylamide gels and agarose Blue G-250 (CBBG) absorbance spectra during titration of gels Brilliant Blue G can be prepared in a deionized water the dye reagent in the absence of protein and its response to solution containing the following: 0.1% (w/v) Brilliant different polyamino acids. The CBBG dye reagent can exist in Blue G, 25% (v/v) methanol, and 5% (v/v) acetic acid. three forms:

• A Cation (red form with a maximum absorbance at 470 nm), • A Neutral Form (green with maximum at 650 nm), • An Anionic Form (blue with maximum at 595 nm).

page 1 Procedure for Staining IEF Gels 3. Bradford, M.M., A Rabid and Sensitive Method for the Quantitation 1. Rinse the gel several times with deionized water. of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem., 72: 248 - 254, (1976).

2. Incubate in staining solution for 4. Matejovicova, M., et al., Effect of Vanadate on Protein about one hour at room temperature. Determination by the Coomassie Brilliant Blue Microassay Procedure. Anal. Biochem., 245(2): 252 - 254, (1997).

3. Prepare the destaining solution by combining 5. Samsonoff, C., et al., The Use of Coomassie Brilliant Blue for Critical 750 ml of methanol, 150 ml of glacial acetic Micelle Concentration Determination of Detergents. Journal of Colloid acid, and 2.1 liters of deionized water. and Interface Science, 109(2): 325 - 329, (1986).

4. Incubate the gel with the destaining solution until Coomassie is a tradename of Imperial Chemical Industries PLC (ICI; London, UK). the gel is clear, changing the solution frequently. Serva Blue G is a tradename of SERVA Electrophoresis GmbH (Heidelberg, Germany) The times for staining and destaining will vary with the size of the gel and other factors.

A solution of 0.4% (w/v) Brilliant Blue G in 3.5% (w/v) perchloric acid may be used to stain PAGE, IEF, and SDS-PAGE gels. This solution may be used as follows:

Procedure for “native PAGE” and IEF gels: 1. Rinse gel for 10 to 30 seconds in deionized water.

2. Incubate gel in staining solution for 30 to 60 minutes.

3. Rinse the gel with deionized water. A destaining step is not required.

Procedure for SDS-PAGE gels: 1. Rinse the gel for 10 to 30 seconds in deionized water.

2. Fix the gel for 30 minutes in fixing solution. The fixing step is required to remove the SDS from the gel.

3. Rinse the gel in several changes of deionized water to completely remove the fixing solution.

4. Incubate the gel in staining solution for 30 to 60 minutes.

5. Rinse the gel with deionized water. A destaining step is not required.

PRECAUTIONS & DISCLAIMER

This product is solely for Research and Development use. It is not for drug, household, or any other uses. Please review the Safety Data Sheet for information regarding risks and safe handling practices.

REFERENCES

1. Green, F.J., The Sigma-Aldrich Handbook of Stains, Dyes & Indicators, p. 151, (1990).

2. Dawson, R.M.C., Data for Biochemical Research, 3rd ed., p. 543, (1986).

NBC warrants that its products will conform with the information provided in this information sheet, though the Purchaser must determine the suitability of the products for their particular usage needs. Additional terms and conditions may apply. MB1131 • 03/12/12 • Rev. 1 page 2