Hi all! I'll be touching on an assay that should be familiar to all of us ---- Bradford Assay.

Bradford Protein Assay is actually a simple and accurate procedure that is used to determine the concentration of protein in solutions.

Bradford assay is a protein determination method that involves the binding of Coomassie Brillant Blue dye to proteins. The dye exists in 3 forms : cationic (red), neutral (green) and anionic (blue). The dye is usually in the protonated red cationic form. However, when the dye binds to protein, it is concverted to a stabe unprotonated blue form. It is this blue dye form that is detected at 595 nm in the assay using an ELISA plate spectrophotometer.

Usually a standard bovine serum albumin (BSA) (2mg/ml) is used to plot the standard curve that will be used to extrapolate the protein concentration of the samples. Various concentrations of the standards BSA will be prepared as follows:

Concurrently, the protein samples are also diluted, usually in a 20x dilution. Once these are prepared, the tubes are then vortex to allow the well mixing and centrifuged to bring down all the proteins.

For my experiments, both the standards and samples are pipetted into a 96-well plate in triplicates, which means that for each standard or sample, there will be 3 wells. 250ul of Coomassie Brillant Blue dye aka Bradford reagent is then added in. Take into consideration that this step is time critical because bradford reagents are light sensitive. Therefore, very often, an aluminium foil will be used to cover the 96-well plate after the addition of the reagents.

When the absorbance are given by the spectrophotometer, a standard graph of absorbance value against protein concentration is plotted. An example of the graph is shown :

With this standard curve, the protein concentration can be extrapolated.

One thing to note when plotting the graph is the R-squared value. The desired R-squared value is 1. However, it is not possible to get this value unless there are only 2 standards. Therefore, for my lab, as long as the R-squared value is greater than 0.99, the graph is accepted and the protein concentration that is extrapolated from this graph is said to be reliable and accurate. If the desired R-squared value of greater than 0.99 cannot be achieved, the assay will be carried out again.


Thats all for bradford assay! Its just sweet and simple! Hope u guys understand! Feel free to ask me any qns!

Take care and see ya all in 2 weeks time!

Charmaine
TG01