2: Bradford Assay
- Page ID
- 146483
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)This assay assesses the concentration of protein in a sample by measuring the absorbance shift from 465 nm (unbound dye) to 595 nm (dye bound to protein) using a spectrophotometer. As Coomassie Brilliant Blue G-250 dye binds to protein, the anionic binding results in a change from a brown/red color to a blue color which can be read by a plate reader to quantify the amount of sample at 595 nm after incubation. The more blue the more protein!
Also known as
Bradford Protein Assay, commercial names (i.e. Bio-Rad protein assay)
Similar techniques: BCA (Bicinchoninic acid) Assay
Samples needed
Samples for this assay can be any kind of solution where you want to measure protein content. Some common forms are cell lysate, purified protein, and plasma.
Method
The experimental protocol for this method is actually quite simple; protein samples are incubated with Coomassie Brilliant Blue dye for around 5-10 minutes in either a cuvette or a multiwell plate and then the absorbance is read by a spectrophotometer to assess protein binding.
To translate these readings into meaningful data you will first need to create a standard curve based on dilutions of your known protein concentrations which will be used to for comparison with your unknown sample absorbances for a final concentration measurement.
Figure 1. Hypothetical workflow, description in text. [Image description]Controls
To properly run this assay you need two controls: a blank (a baseline solution with no protein) and a set of protein standards (samples with known protein concentrations much like a ladder in gel electrophoresis). The blank is used to establish a baseline for your sample buffer and the known protein dilutions are used to generate a standard curve to compare unknown absorbances.
Interpretation
Figure 2. Standard curve results of wheat protein fractions. Paper figure reads: “Linear graphs obtained with the Bradford assay using BSA (blue) and the purified wheat protein fractions Albumin–Globulin (brown), Gliadin (red) and Glutenin (green) as calibrants. Slope and y-intercept from the four derived equations. All values were measured in four replicates.” “Figure 1” of Rekowski et al.[1] [Image description]These researchers used the absorbance of their known proteins (BSA, albumin-globulin, gliadin, glutenin), as control standards for their proteins of interest. BSA is the most commonly used protein standard but researchers may choose others, as shown above, if they are closer in composition to their unknowns of interest. The lines of best fit in these standards are then compared to assess protein concentration in their experimental groups.
Image Descriptions
Figure 1 image description:
The image on the left shows an example of a multiwell plate with a blue gradient covering the left half as the protein standard dilutions and a range of colors (blue, orange, brown, red) on the right side as experimental unknown samples. The right side shows an example of a standard curve with corresponding line of best fit. The y axis is absorbance at 595 nm and the x axis is amount of protein in ug. Below the standard curve, the equation of the line is used to assess the hypothetical unknown sample with the sample absorbance in place of the y value. ↵
Figure 2 image description:
This image shows multiple Bradford standard curves, ug of protein vs. absorbance at 595 nm, with corresponding equations. The lines with slopes from greatest to least are composed using standardized protein solutions of BSA (bovine serum albumin), albumin-globulin, gliadin, and glutenin.↵
Author
Emilie Jones, Tufts University
1. Rekowski, A., Langenkämper, G., Dier, M., Wimmer, M. A., Scherf, K. A., & Zörb, C. (2021). Determination of soluble wheat protein fractions using the Bradford assay. Cereal Chemistry, 98(5), 1059–1065. ↵