A common method of measuring the concentration of proteins in a sample. The binding of a dye to proteins causes a shift in the dye’s absorbance maximum, allowing for quantification.
The Bradford Assay is a colorimetric (relying on color change) protein quantification method. At the heart of this assay is the Coomassie Brilliant Blue G-250 dye. In its unbound form, this dye is reddish-brown and absorbs light at 465 nm. However, when it binds to protein, primarily through arginine and lysine residues, the dye undergoes a color change to blue and its maximum absorbance shifts to 595 nm. This change in color and absorbance is directly proportional to the protein concentration, allowing for quantification.
After each purification step, it’s essential to determine the concentration of the purified protein to assess the yield and purity. The Bradford Assay provides a quick and reliable method to quantify the protein in these fractions. It helps researchers decide which fractions to pool and aids in determining the efficiency and success of the purification process.
Before using the lysate for downstream applications, it’s crucial to know its protein concentration. This ensures that equal amounts of protein are used across samples, allowing for consistent and comparable results. The Bradford Assay is commonly used to determine the total protein content in these lysates.
For accurate results, it’s essential that each well in an SDS-PAGE gel receives an equal amount of protein. This ensures that when comparing bands across lanes, any differences observed are due to the experimental conditions and not unequal loading. The Bradford Assay is used to adjust the protein concentrations of samples so that equal amounts of protein are loaded into each well.
This is the most common method and involves creating a calibration curve using known concentrations of a standard protein, typically Bovine Serum Albumin (BSA) or gamma globulin.
A quicker alternative to the standard curve method. It involves estimating the protein concentration of an unknown sample using a single known concentration of a standard protein.
This method adapts the Bradford Assay for high-throughput analysis using microplates, allowing for the simultaneous measurement of multiple samples.