0 µg/mL to 2000 µg/mL, often making dilutions of a sample necessary before analysis. In making these dilutions, error in one dilution is compounded in further dilutions resulting in a linear relationship that may not always be accurate. Basic conditions and detergents, such as SDS, can interfere with the dye's ability to bind to the protein through its side chains. However, there are some deterd protein, it is possible that the concentration measured will be inaccurate. The reagents in this method tend to stain the test tubes. Same test tubes cannot be used since the stain would affect the absorbance reading. This method is also time sensitive. When more than one solution is tested, it is important to make sure every sample is incubated for the same amount of time for accurate comparison. It is also inhibited by the presence of detergents, although this problem can be alleviated by the addition of cyclodextrins to the assay mixture. Much of the non-linearity stems from the equilibrium between two different forms of the dye which is perturbed by adding the protein. The Bradford assay linearizes by measuring the ratio of the absorbances, 595 over 450 nm. This modified Bradford assay is approximately 10 times more sensitive than the conventional one. The Coomassie Blue G250 dye used to bind to the proteins in the original Bradford method readily binds to arginine and lysine groups of proteins. This is a disadvantage because the preference of the dye to bind to these amino acids can result in a varied response of the assay between different proteins. Changes to the original method, such as increasing the pH by adding NaOH or adding more dye have been made to correct this variation. Although these modifications result in a less sensitive assay, a modified method becomes sensitive to detergents that can interfere with sample. Sample Bradford procedure Materials Lyophilized bovine plasma gamma globulin Coomassie brilliant blue 1 0.15 M NaCl Spectrophotometer and cuvettes or a mobile smartphone camera (RGBradford method). Micropipettes Procedure (Standard Assay, 20-150 µg protein; 200-1500 µg/mL) Prepare a series of standards diluted with 0.15 M NaCl to final concentrations of 0 (blank = No protein), 250, 500, 750 and 1500 µg/mL. Also prepare serial dilutions of the unknown sample to be measured. Add 100 µL of each of the above to a separate test tube (or spectrophotometer tube if using a Spectronic 20). Add 5.0