text: The Bradford assay, a colorimetric protein assay, is based on an absorbance shift of the dye  Coomassie brilliant blue G-250.  The Coomassie brilliant blue G-250 dye exists in three forms: anionic (blue), neutral (green), and cationic (red). Under acidic conditions, the red form of the dye is converted into its blue form, binding to the protein being assayed. If theres no protein to bind, then the solution will remain brown. The dye forms a strong, noncovalent complex with the proteins carboxyl group by van der Waals force and amino group through electrostatic interactions. During the formation of this complex, the red form of Coomassie dye first donates its free electron to the ionizable groups on the protein, which causes a disruption of the proteins native state, consequently exposing its hydrophobic pockets. These pockets in the proteins tertiary structure bind non-covalently to the non-polar region of the dye via the first bond interaction (van der Waals forces) which position the positive amine groups in proximity with the negative charge of the dye. The bond is further strengthened by the second bond interaction between the two, the ionic interaction. When the dye binds to the protein, it causes a shift from 465 nm to 595 nm, which is why the absorbance readings are taken at 595 nm.
  The cationic (unbound) form is green / red and has an absorption spectrum maximum historically held to be at 465 nm. The anionic bound form of the dye which is held together by hydrophobic and ionic interactions, has an absorption spectrum maximum historically held to be at 595 nm. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample.
  Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemical compounds such as sodium, potassium or even carbohydrates like sucrose, that may be present in protein samples.  An exception of note is elevated concentrations of detergent. Sodium dodecyl sulfate (SDS), a common detergent, may be found in protein extracts because it is used to lyse cells by disrupting the membrane lipid bilayer and to denature proteins for SDS-PAGE. While other detergents interfere with the assay at high concentration, the interference caused by SDS is of two different modes, and each occurs at a different concentration. When SDS concentrations are below critical micelle concentration (known as CMC, 0.00333%W/V to 0.0667%) in a Coomassie dye solution, the detergent tends to bind strongly with the protein, inhibiting the protein binding sites for the dye reagent. This can cause underestimations of protein concentration in solution. When SDS concentrations are above CMC, the detergent associates strongly with the green form of the Coomassie dye, causing the equilibrium to shift, thereby producing more of the blue form. This causes an increase in the absorbance at 595 nm independent of protein presence.
  Other interference may come from the buffer used when preparing the protein sample. A high concentration of buffer will cause an overestimated protein concentration due to depletion of free protons from the solution by conjugate base from the buffer. This will not be a problem if a low concentration of protein (subsequently the buffer) is used.
  In order to measure the absorbance of a colorless compound a Bradford assay must be performed. Some colorless compounds such as proteins can be quantified at an Optical Density of 280 nm due to the presence of aromatic rings such as Tryptophan, Tyrosine and Phenylalanine but if none of these amino acids are present then the absorption cannot be measured at 280 nm.