Protein quantification is integral to many protein isolation, detection and analysis workflows. Bio-Rad offers colorimetric assays for the quantification of proteins based on well-document Bradford and Lowry assays. Our Bradford protein assays are popular, easy-to-use protein quantification kits that enable researchers to quantitate a wide range of protein concentrations quickly and accurately. Check out the kits below to find out more information about our Bradford protein assays.
How the Bradford Protein Assay Works
The Bradford protein assay is a time-tested colorimetric assay. When the Bradford reagent (acidified Coomassie Brilliant Blue G-250) binds to proteins, the dye undergoes a color change in the visible spectrum, with the absorbance maximum moving from 470 to 595 nm. The absorbance at 595 nm is then read either in a spectrophotometer or a microplate reader and is directly proportional to the amount of protein bound. The exact protein concentration of the sample is determined by interpolation from a standard curve made by measuring the absorbance of a dilution series of protein standards of known concentrations within the linear response range of the assay. Proteins commonly used as standards include bovine serum albumin (BSA) and bovine γ-globulin (BGG).
There are two types of kits. One contains ready-to-use reagent and prediluted BSA or BGG standards for a simple and quick assay. The other kit provides more flexibility with 5x concentrated reagent and lyophilized BSA or bovine-globulin standards, allowing the user to prepare reagent and standards at any concentration.
All protein assays are subject to interference by certain substances under some conditions. The assay is quite robust and is compatible with many compounds commonly found in protein preparations. A standard Bradford protein assay kit is compatible with the following chemicals:
- Denaturing agents such as sodium thiocyanate, guanidine HCl, urea, and phenol
- Reducing agents such a dithiothreitol and β-mercaptoethanol
- Buffers including HEPES, MES, MOPS, Tris, and phosphate
- Chelating agents such as EDTA and EGTA
- Salts such as NaCl, KCl, and MgCl2
- Tissue culture media such as Eagle's MEM and Hank's salt solution
- Sugars such as sucrose, glucose, and fructose
- DNA and RNA
- Organic solvents such as acetone, methanol, and ethanol
- Detergents such as Triton™ X-100 and deoxycholate, and low concentrations of SDS
Additionally, free amino acids and small peptides (<3 kDa) do not interfere with the assay.
For membrane preparations that have been solubilized with detergents, depending on the type of detergent and the concentration, it may be necessary to dilute the sample to reduce the concentration. If a quick Bradford protein assay kit with a ready-to-use Bradford reagent is used, detergents that interfere will need to be at a lower concentration in the sample than in the standard assay due to the high sample-to-dye ratio. Dilution may also be required in the presence of flavonoids and some basic buffers, as they can also interfere with Bradford protein assays.
Bio-Rad kits include either of two different proteins, BSA or BGG, as standards to construct a standard curve for the relative quantitation of the proteins in the samples. For most determinations of protein concentration, relative values are generally sufficient. BSA is the most commonly used standard for relative protein concentration determination in most laboratories, although the color response of γ-globulin is usually more representative of true concentration for samples that do not have a high albumin content.
If necessary, the accuracy of the standards can be increased by preparing the standard dilutions in the same buffer as the samples. Typically, seven concentrations of standards are prepared to cover the linear range of a Bradford protein assay, ranging from 0.125 to 2 mg/ml.