Staining of proteins in gels with Coomassie G-250 without organic solvent and acetic acid.
In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80 mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staining of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compounds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.
Lawrence
Ann-Marie
AM
Protein Expression and Purification Core Facility, EMBL Heidelberg.
Besir
H Uuml Seyin
HU
D019342:Acetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels / Q000737:chemistry*; D007202:Indicators and Reagents / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012394:Rosaniline Dyes / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*; D014867:Water / Q000737:chemistry
D005782:Gels; D007202:Indicators and Reagents; D011506:Proteins; D012394:Rosaniline Dyes; D012997:Solvents; D014867:Water; C004692:coomassie Brilliant Blue; D019342:Acetic Acid