What is Coomassie r250?
What is Coomassie r250?
Coomassie R-250 and G-250 dyes are two chemical forms of a disulfonated triphenylmethane compound that is commonly used as the basis of stains for detection of proteins in gel electrophoresis and Bradford-type assay reagents for protein quantitation.
How do you make Coomassie blue staining r250?
Prepare the staining solution containing 0.1% Coomassie R-250 in 40% ethanol, 10% acetic acid. After electrophoresis, incubate 1 or 2 gels in a staining container containing 100 ml Coomassie Blue R-250 staining solution.
How does Coomassie Brilliant Blue 250 stain protein?
CBB R-250 and the dimethyl derivative CBB G-250 are disulfonated triphenylmethane dyes that stain protein bands bright blue. The dyes bind via electrostatic interaction with protonated basic amino acids (lysine, arginine, and histidine) and by hydrophobic associations with aromatic residues.
What does Coomassie blue staining show?
Description. Coomassie blue dyes are a family of dyes commonly used to stain proteins in SDS-PAGE gels. This treatment allows the visualization of proteins as blue bands on a clear background.
Does Coomassie stain all proteins?
The most common used protein stain is Coomassie Blue staining, which is based on the binding of Coomassie Brilliant Blue, which binds non-specifically to virtually all proteins.
What is the difference between Coomassie r250 and g250?
Like R-250, Coomassie G-250 (also known as colloidal Coomassie dye) also offers relatively high sensitivity and involves a simple protocol. However, G-250 offers a faster staining protocol and eliminates the need for destaining the gel (you can easily visualize the protein bands against the light amber background).
Can I Destain overnight?
Expose the gel in staining solution overnight and destain the gel by changing water frequently.
Can you Coomassie stain overnight?
Note: Gel can be stained for up to 3 hours, but after 3 hours, sensitivity will decrease. If you need to leave the gel overnight in the stain, add 2 ml of 20% NaCl (w/v) in water for every 20 ml of stain. This procedure will not affect sensitivity.
How much protein should I load for Coomassie?
For a mini gel being stained with Coomassie brilliant blue R-250, load a maximum of 50 g for a complex sample. For a sample with only one or a few bands, load about 1 g per band. If your protein of interest is in low abundance, you may load up to 100 g of protein, but the resolution of the gel may be poor.
How does Coomassie G-250 interact with proteins?
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.
How much protein do you need for Coomassie stain?
For standard Coomassie staining, I generally load about 2ug of a singular purified protein per well. You should get a crisp dark band. In my experience, if you go much over 5ug your band will start to become more blob and less band.
How do you use Coomassie gel stain?
Rinse gel with water. Add ~200 ml protein gel destain. Microwave ~45 sec until the solution just starts to boil. Incubate with gentle shaking at room temp for ~15 min.
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