The Bradford Protein Assay, often associated with Bovine Serum Albumin (BSA) due to its frequent use as a protein standard, relies on the binding of the Coomassie Brilliant Blue G-250 dye to proteins. The primary components of the Bradford assay are:

1. Coomassie Brilliant Blue G-250 Dye: This is the central component of the assay. In its unbound state (i.e., not bound to proteins), the dye is brownish-green. Upon binding to proteins, it turns blue. The intensity of this blue coloration is indicative of the amount of protein present.
2. Acid Solution: The dye is dissolved in an acidic solution, which helps in maximizing the dye’s response to proteins. Phosphoric acid or other acids can be used to maintain the acidic environment. The acidic environment ensures the dye is in its protonated, neutral form, which is necessary for the subsequent binding to protein and transition to the anionic blue form.
3. Protein Sample: This could be a lysate from cells or tissues, purified proteins, or any other sample that contains proteins to be quantified.
4. Protein Standard: Often Bovine Serum Albumin (BSA) or another well-characterized protein is used as a standard. A series of known concentrations of this standard protein is prepared and subjected to the assay alongside the unknown samples. This creates a standard curve against which the protein concentration of unknown samples can be compared and determined.
5. Buffer: Sometimes, a buffer might be present, especially if you’re working with lysates or samples that have been prepared in a specific buffer solution. However, it’s essential to note that some buffer components might interfere with the assay, so it’s always a good idea to test and calibrate the assay under your specific conditions.
6. Distilled Water: Used to prepare dilutions or to serve as a blank in spectrophotometric measurements.

When performing the assay:

1. The dye reagent is added to both the protein sample and the protein standard solutions.
2. The mixtures are allowed to incubate for a short duration, during which the color change occurs.
3. The absorbance of each sample is then read at 595 nm using a spectrophotometer. A blank sample, consisting of the dye reagent and any buffer or solvent without protein, is also read to subtract any background absorbance.
4. Using the known concentrations and absorbance values from the protein standards, a standard curve is plotted, which can then be used to determine the protein concentration of the unknown samples.

It’s essential to always follow the specific protocol associated with your Bradford reagent, as there can be slight variations based on the manufacturer or specific laboratory modifications.