Bradford Protein Assay Protocol

Materials Provided

• 100µg/ml Bovine Serum Albumine (BSA)

• Bradford solution

Step 1

Assemble Bradford assay in this order:

• protein standard/sample protein: up to 800µl

• dH₂O: to a total volume of 800µl

• Bradford reagent: 200µl

Bradford reagent is added last bc it starts the color-forming reaction (binds to protein immediately).

• adding it last ensures all samples start reacting at the same time

Step 2

• mix well (cuvettes should be one color after mixing)

• incubate for 2 mins at room temp

• read OD595nm within 60 min

Step 3

• to produce a standard curve for protein mass, perform a series of assays containing 0, 1, 2, 3, 5 and 10 µg of BSA

• plot OD595nm versus µg BSA standard

Step 4

Compare values for your sample to the standard curve

Theory

Bradford Assay

• colorimetric protein assay, and is based on absorbance shift of Coomassie blue dye

• a rapid and accurate method for the estimation of protein concentration

• less subject to interference by various chemical compounds (e.g. Na, K, carbohydrates) that may be preset in protein samples

Coomassie Blue Dye

• exists in three forms: anionic (blue), neutral (green) and cationic (red)

• under acidic conditions, the red form of the dye is converted into the blue form upon binding to proteins thru noncovalent interactions (VDW and ionic)

• his binding exposes hydrophobic regions of the protein and stabilizes the dye–protein complex.

• The color change causes an absorbance shift from 465 nm to 595 nm, so protein concentration is measured at 595 nm.

Limitation of Coomassie

• binds most readily to arginine and lysine residues of proteins

• this specificity can lead to variation in the response of the assay to different proteins

• this is not as problematic in larger proteins, because the amino acid content will average to some degree

How does SDS interfere with the Bradford assay?

• The Bradford assay is generally resistant to many compounds, but SDS (a detergent) can interfere

• below critical micelle concentration (CMC): SDS binds to proteins and blocks dye-binding sites → leads to underestimation of protein concentration

• above CMC: SDS interacts with Coomassie dye and shifts equilibrium to the blue form even w/o protein → leads to overestimation of protein concentration

How does high buffer concs interfere with the Bradford assay?

High buffer concentrations can also overestimate protein levels by altering proton availability.