Bradford protein assay
Encyclopedia
The Bradford protein assay is a spectroscopic
analytical procedure used to measure the concentration of protein
in a solution. It is subjective, i.e., dependent on the amino acid composition of the measured protein. The Bradford protein assay was developed by Marion M. Bradford
.
, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its bluer form to bind to the protein being assayed. During the formation of this complex, two types of bond interaction take place: the red form of Coomassie dye first donates its free electron to the ionizable groups on the protein, which causes a disruption of the protein's native state, consequently exposing its hydrophobic pockets. These pockets on the protein's tertiary structure
bind non-covalently to the non-polar region of the dye via van der Waals force
s, positioning the positive amine groups in proximity with the negative charge of the dye. The bond is further strengthened by the ionic interaction between the two. The binding of the protein stabilizes the blue form of the Coomassie dye; thus the amount of the complex present in solution is a measure for the protein concentration, and can be estimated by use of an absorbance reading.
The (bound) form of the dye has an absorption spectrum maximum historically held to be at 595 nm
. The cationic (unbound) forms are green or red. The binding of the dye to the protein stabilizes the blue anionic form. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample.
Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemicals that may be present in protein samples. An exception of note is elevated concentrations of detergent
. Sodium dodecyl sulfate
(SDS), a common detergent, may be found in protein extracts because it is used to lyse cells by disrupting the membrane lipid bilayer. While other detergents interfere with the assay at high concentration, the interference caused by SDS is of two different modes, and each occurs at a different concentration. When SDS concentrations are below critical micelle concentration (known as CMC, 0.00333%W/V to 0.0667%) in a Coomassie dye solution, the detergent tends to bind strongly with the protein, inhibiting the protein binding sites for the dye reagent. This can cause underestimations of protein concentration in solution. When SDS concentrations are above CMC, the detergent associates strongly with the green form of the Coomassie dye, causing the equilibrium to shift, thereby producing more of the blue form. This causes an increase in the absorbance at 595 nm independent of protein presence.
Other interference may come from the buffer used when preparing the protein sample. A high concentration of buffer will cause an overestimated protein concentration due to depletion of free protons from the solution by conjugate base from the buffer. This will not be a problem if a low concentration of protein (subsequently the buffer) is used.
It is also inhibited by the presence of detergents.
Much of the non-linearity stems from the equilibrium between two different forms of the dye which is perturbed by adding the protein. The Bradford assay linearizes by measuring the ratio of the absorbances, 595 over 450 nm. This modified Bradford assay is approximately 10 times more sensitive than the conventional one. (Zor & Selinger, 1995)
Spectroscopy
Spectroscopy is the study of the interaction between matter and radiated energy. Historically, spectroscopy originated through the study of visible light dispersed according to its wavelength, e.g., by a prism. Later the concept was expanded greatly to comprise any interaction with radiative...
analytical procedure used to measure the concentration of protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
in a solution. It is subjective, i.e., dependent on the amino acid composition of the measured protein. The Bradford protein assay was developed by Marion M. Bradford
Marion M. Bradford
Marion M. Bradford is a scientist who developed and patented the Bradford protein assay, a method to quickly quantify the amount of protein in a sample. Bradford's paper describing the method is among the most cited scholarly articles of all time.-References:...
.
Principle
The Bradford assay, a colorimetric protein assayAssay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its bluer form to bind to the protein being assayed. During the formation of this complex, two types of bond interaction take place: the red form of Coomassie dye first donates its free electron to the ionizable groups on the protein, which causes a disruption of the protein's native state, consequently exposing its hydrophobic pockets. These pockets on the protein's tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
bind non-covalently to the non-polar region of the dye via van der Waals force
Van der Waals force
In physical chemistry, the van der Waals force , named after Dutch scientist Johannes Diderik van der Waals, is the sum of the attractive or repulsive forces between molecules other than those due to covalent bonds or to the electrostatic interaction of ions with one another or with neutral...
s, positioning the positive amine groups in proximity with the negative charge of the dye. The bond is further strengthened by the ionic interaction between the two. The binding of the protein stabilizes the blue form of the Coomassie dye; thus the amount of the complex present in solution is a measure for the protein concentration, and can be estimated by use of an absorbance reading.
The (bound) form of the dye has an absorption spectrum maximum historically held to be at 595 nm
Nanometre
A nanometre is a unit of length in the metric system, equal to one billionth of a metre. The name combines the SI prefix nano- with the parent unit name metre .The nanometre is often used to express dimensions on the atomic scale: the diameter...
. The cationic (unbound) forms are green or red. The binding of the dye to the protein stabilizes the blue anionic form. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample.
Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemicals that may be present in protein samples. An exception of note is elevated concentrations of detergent
Detergent
A detergent is a surfactant or a mixture of surfactants with "cleaning properties in dilute solutions." In common usage, "detergent" refers to alkylbenzenesulfonates, a family of compounds that are similar to soap but are less affected by hard water...
. Sodium dodecyl sulfate
Sodium dodecyl sulfate
Sodium dodecyl sulfate , sodium laurilsulfate or sodium lauryl sulfate is an organic compound with the formula CH311OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products...
(SDS), a common detergent, may be found in protein extracts because it is used to lyse cells by disrupting the membrane lipid bilayer. While other detergents interfere with the assay at high concentration, the interference caused by SDS is of two different modes, and each occurs at a different concentration. When SDS concentrations are below critical micelle concentration (known as CMC, 0.00333%W/V to 0.0667%) in a Coomassie dye solution, the detergent tends to bind strongly with the protein, inhibiting the protein binding sites for the dye reagent. This can cause underestimations of protein concentration in solution. When SDS concentrations are above CMC, the detergent associates strongly with the green form of the Coomassie dye, causing the equilibrium to shift, thereby producing more of the blue form. This causes an increase in the absorbance at 595 nm independent of protein presence.
Other interference may come from the buffer used when preparing the protein sample. A high concentration of buffer will cause an overestimated protein concentration due to depletion of free protons from the solution by conjugate base from the buffer. This will not be a problem if a low concentration of protein (subsequently the buffer) is used.
Disadvantages
The Bradford assay is linear over a short range, typically from 0 µg/ml to 2000 µg/ml, often making dilutions of a sample necessary before analysis.It is also inhibited by the presence of detergents.
Much of the non-linearity stems from the equilibrium between two different forms of the dye which is perturbed by adding the protein. The Bradford assay linearizes by measuring the ratio of the absorbances, 595 over 450 nm. This modified Bradford assay is approximately 10 times more sensitive than the conventional one. (Zor & Selinger, 1995)
Materials
- Lyophilized bovine plasma gamma globulin or bovine serum albuminBovine serum albuminBovine serum albumin is a serum albumin protein derived from cows. It is often used as a protein concentration standard....
(BSA) - Coomassie Brilliant Blue 1
- 0.15 M NaCl
- Spectrophotometer and tubes
- Micropipettes
Procedure (Standard Assay, 20-150 µg protein; 200-1500 µg/ml)
- Prepare a series of protein standards using BSA diluted with 0.15 M NaCl to final concentrations of 0 (blank = NaCl only), 250, 500, 750 and 1500 µg BSA/mL. Also prepare serial dilutions of the unknown sample to be measured.
- Add 100 µL of each of the above to a separate test tube (or spectrophotometer tube if using a Spec 20).
- Add 5.0 µL of Coomassie Blue to each tube and mix by vortex, or inversion.
- Adjust the spectrophotometer to a wavelength of 595 nm, and blank using the tube which contains 0 BSA.
- Wait 5 minutes and read each of the standards and each of the samples at 595 nm wavelength.
- Plot the absorbance of the standards vs. their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.
Procedure (Micro Assay, 1-10 µg protein/mL)
- Prepare standard concentrations of BSA of 1, 5, 7.5 and 10 µg/mL. Prepare a blank of NaCl only. Prepare a series of sample dilutions.
- Add 100 µL of each of the above to separate tubes (use microcentrifuge tubes) and add 1.0 mL of Coomassie Blue to each tube.
- Turn on and adjust a spectrophotometer to a wavelength of 595 nm, and blank the spectrophotometer using 1.5 mL cuvettes.
- Wait 2 minutes and read the absorbance of each standard and sample at 595 nm.
- Plot the absorbance of the standards vs. their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.
Alternative assays
Alternative protein assays include- Ultraviolet–visible spectroscopy
- Biuret protein assay
- Lowry protein assayLowry protein assayThe Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques. It is...
- Bicinchoninic acid protein assayBicinchoninic acid assayThe bicinchoninic acid assay , also known as the Smith assay, after its inventor, Paul K. Smith at the Pierce Chemical Company, is a biochemical assay for determining the total level of protein in a solution , similar to Lowry protein assay, Bradford protein assay or biuret reagent...
- Amido black protein assay
- o-phthalaldehyde protein assay
- Nano-orange