The Quick Start™ Bradford protein assay is a fast, robust and simple one-step method for measuring protein concentrations samples in a variety of solutions. The Bradford method (Anal. Biochem. (1976) 72, 248-254) measures the absorbance shift of the red cationic form of Coomassie Blue G-250 to the blue stable anionic form when bound to arginine and hydrophobic amino acids in proteins.
The Quick Start™ Bradford Protein Assay Kit contains 1 liter dye solution (includes methanol and phosphoric acid) and either five aliquots (2 mg x 2 ml each) of bovine serum albumin or bovine gamma-globulin standards, or two aliquots (2 ml each) of pre-diluted standard sets: 2, 1.5, 0.75, 0.5, 0.25, 0.125 mg/ml in screwcap vials. The kits are stable for 1 year at 4°C since the standards are diluted in 0.9% NaCl/0.05% NaN3. Standard assays are performed as 5 ml (sample 100 µl), 1 ml (sample 20 µl) or 250 ul (sample 5 µl, in microtiter plate) assays; microassays are performed with equal volumes of dye and samples either with 2 ml in tubes or 150 µl in a microtiter plate). The protein concentrations can be measured after 5 minutes or up to 1 h; measurements are taken at 595 nm (580 - 610 nm) in a spectrophotometer or microplate reader. The Quick Start™ Bradford Protein Assay Kit includes enough reagents for 200 standard tube assays or 4,000 microplate assays.
The assay is compatible with a wide range of reagents, such as organic solvents (acetone, acetonitrile, methanol), some detergents (CHAPS, desoxycholate, octylglucoside), most commonly used buffers (PBS, TRIS, Hepes), culture media (Eagles MEM), salt solutions (NaCl2, MgCl2, KCl), sugars (sucrose, glucose) and denaturating reagents (DTT, 2-mercaptoethanol, urea). The instruction manual provides a detailed protocol of the procedure, a complete list of compatible reagents as well as a troubleshooting section. The microassay is compatible with lower concentrations of reagent (1/25 of the amount of reagent required for the standard assay) listed in the table due to a higher sample-dye volume. Interference of any reagents with the protein of interest should be tested or the buffer of the unknown samples should be included in the standards when preparing the dilutions. Proteins with molecular weights lower than 3-5 kDa cannot be detected with the assay.
Measuring samples from a variety of tissues and cell lysates, I especially liked using the microplate standard assay. We routinely used the assay with the bovine gamma globulin standard (in duplicate or triplicate), which has a wider detection range than BSA and resembled more closely one of our protein lysates, an Fc-construct of a recombinant protein. The microplate assay is especially useful when multiple samples have to be measured at the same time for accurate comparison (e.g. different time points after cell stimulation to detect changes in phosphorylation or expression levels in a signaling pathway). In the microplate assay, a multi-channel pipettor can be used to mix the dye solution with the samples in order to speed up the pipetting process and handle less of the hazardous solution. Care has to be taken that equal volumes are pipetted and no spills occur when mixing too quickly.
It is easy to measure the assay in a microplate reader, which can be mostly programmed to subtract the blank value, set up the standard curve and calculate directly the concentrations of the samples. When using the assay with buffers containing reagents not listed in the compatibility table of the protocol, it is better to set up a standard curve with the buffer and another one with a listed buffer to make sure that the color development can be measured with the method. (Inclusion of incompatible detergents or reagents can change the expected blue color into a variety of different colors).
The Quick Start™ Bradford Protein Assay Kits are a fast, reliable and easy way to determine protein concentrations in multiple samples even when different protein concentrations are expected in the range of 1.25 - 20 µg/ml (microplate assay). The instruction manual provides all the information needed to perform the assay, as well as suggestions for standard dilutions and a troubleshooting guide.