How do I prepare a solution of Coomassie R-250 dye?
How do I prepare a solution of Coomassie R-250 dye? Add 100 mL of glacial acetic acid to 450 mL. Dissolve 3 g of Coomassie R-250 dye in 450 mL methanol. Mix the acetic acid and methanol solutions.
What is the difference between R250 and G250 Coomassie Brilliant Blue G 250?
The more popular is Coomassie R250 (Reddish tinted blue) for electrophoresis (more sensitive: can detect as little as 0.1 µg of protein), and Coomassie G250 (Greenish tinted blue) for protein assay in solutions (because it is more convenient – soluble).
Can you Coomassie stain overnight?
Stain for 1-4 hours or overnight at room temperature with gentle shake. Coomassie blue stain solution can be reused for serveral times.
How do you make a Coomassie stain?
To prepare 1 l of a staining solution, dissolve 1 g of Coomassie R250 to 300 ml of methanol. Then add 650 ml of MQ water and 50 ml of acetic acid. Stir the solution on a magnetic stirrer for 2 h. The solution can be filtered through a Whatman No.
How does Coomassie Brilliant Blue G-250 bind to a protein?
Coomassie R-250 is the more commonly used and sensitive of the two. In the staining reaction, the Coomassie dye binds to proteins through ionic interactions between sulfonic acid groups and positive protein amine groups through Van der Waals attractions.
What does the G-250 in the Coomassie Brilliant Blue G-250 mean?
The suffix “R” in the name of Coomassie Brilliant Blue R-250 is an abbreviation for Red as the blue colour of the dye has a slight reddish tint. For the “G” variant the blue colour has a more greenish tint. The “250” originally denoted the purity of the dye.
How does Coomassie Brilliant Blue G 250 bind to a protein?
What does the G 250 in the Coomassie Brilliant Blue G 250 mean?
How long does a coomassie stain take?
To stain, immerse gel in above solution. Bands will begin to appear within 15 minutes. Intensity and sensitivity will continue to improve for several hours. Staining solution is stable for 2 – 3 weeks @ 25°C.
How long Coomassie staining is enough?
Microwave on high power for 40 seconds to 1 minute (until the Coomassie Stain boils). Incubate the gel in the Coomassie stain for 5 to 10 minutes on a rocking table. If you did not microwave the Coomassie/gel, incubate for at least 1 hour.
What stains Coomassie?
Coomassie blue dyes are a family of dyes commonly used to stain proteins in SDS-PAGE gels. The gels are soaked in dye, and excess stain is then eluted with a solvent (“destaining”). This treatment allows the visualization of proteins as blue bands on a clear background.
How to stain proteins in gel with Coomassie G-250?
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.
How to remove Coomassie Blue G-250 stain from glass?
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.
Can you use Coomassie G-250 without acetic acid?
Staining of proteins in gels with Coomassie G-250 without organic solvent and acetic acid
What is the absorption wavelength of G250 dye?
At a pH of less than 0 the G250 dye caries all three nitrogen atoms with a positive charge, it has a red colour with an absorption maximum at a wavelength of 465 nm.