How does Coomassie brilliant blue g250 work?
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.
Can one use Commassie brilliant blue R-250 in Bradford’s reagent?
The Bradford reagent, comprised of the Coomassie Brilliant Blue G-250 dye, methanol, and phosphoric acid, has been traditionally used for quantifying proteins. Use of this reagent in the Bradford assay relies on the binding of the Coomassie Blue G-250 dye to proteins.
How do you Destain Coomassie gel?
Destain the gel by soaking for at least 2 hours in 10% acetic acid, 50% methanol, and 40% H2O with at least two changes of this solvent. If the gel still has a Coomassie Blue background then continue destaining until the background is nearly clear.
What is the difference between Coomassie R250 and g250?
G-250: Which One Should You Use? Basically, both R-250 (R signifies the slightly reddish tint in the blue color of the dye) and G-250 (G signifies the greenish tint in the dye) have relatively high sensitivity and allow for easy detection (they develop intensely colored complexes upon binding with protein molecules).
Why does Bradford reagent turn blue?
In the absence of protein, when the dye is red, Bradford reagent has an absorbance maximum (Amax) of 470 nm. In the presence of protein, the change to the anionic blue form of the dye shifts the Amax to 595 nm.
Is Coomassie Blue the same as bromophenol blue?
Coomassie is used for protein staining after SDS/PAGE running and bromophenol blue is used as a migration front indicator (and pH indicator too) during running. BB is part of the loading buffer.
Is Brilliant blue the same as Coomassie?
Coomassie stains (also known as Coomassie Blue or Coomassie Brilliant Blue) are the most popular anionic protein dyes used in visualizing proteins in SDS-PAGE gels. While they may not be as sensitive as fluorescent and silver stains, these dyes provide good quantitative linearity and medium sensitivity.
What is the difference between Coomassie R250 and G250?
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).
How do I increase the accuracy of my Bradford assay?
In different wells, add 100 μl of unknown sample in triplicates. Several concentrations of the unknown sample may be used to increase accuracy. Add 100 μl of the diluted Bradford reagent to all wells. Total volume is 200 μl/well.
How do you use Coomassie Blue?
Microwave for ~45 sec until the solution just starts to boil. Incubate at room temp with gentle shaking for 10-15 min. Heating allows the gel to stain faster. Alternatively, soak gel in stain for 1 hr at room temperature.
Is Brilliant blue the same as bromophenol blue?
Bromophenol blue is used as a front tracking dye, as a pH indicator and doesn’t interact with proteins. Whereas Coomassie brilliant blue will mainly stick to proteins…