Figure 1. Reaction schematic for the Coomassie dye–based Bradford protein assays.
Dye-based protein detection
Bradford assays are dye-binding assays for fast and simple protein quantification. The assay is performed at room temperature and no special equipment is required. Standard and unknown samples are added to pre-formulated Coomassie blue G-250 assay reagent and the resultant blue color is measured at 595 nm following a short room temperature incubation. Bradford protein assays are compatible with most salts, solvents, buffers, thiols, reducing substances, and metal chelating agents encountered in protein samples.
For added ease-of-use, select Bradford assays are now offered with Dilution-Free protein standards that help reduce assay setup time and pipetting errors. Dilution-Free protein standards are a set of prediluted standards packaged in a multichannel pipette-compatible format that enable easy transfer of a full dilution series to your microplate at once.
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Choose the right Bradford protein assay or reagent for your application
 |  |  | |
| Bradford Plus Protein Assay Kit with Dilution-Free BSA Protein Standards | Detergent Compatible Bradford Assay | Bradford Protein Assay | |
|---|---|---|---|
| Features |
|
|
|
| Assay range (sample volume) | 125–1,500 µg/mL (5 µL) Or 1–25 µg/mL (150 µL) | 100–1,500 µg/mL (10 µL) or 2–25 µg/mL (150 µL) | 100–1,500 µg/mL (20 µL) or 1–25 µg/mL (150 µL) |
| Compatible reagents | Buffer salts, metal ions, reducing agents, chelators | Detergents, buffer salts, metal ions, reducing agents, chelators | Buffer salts, metal ions, reducing agents, chelators |
| Incompatibilities | Detergents | >5% detergent solutions | Detergents |
| Incubation time | 10 min | 10 min | 10 min |
| Product size | 950 mL/kit | 450 mL/kit | 950 mL/kit |
| Cat. No. | A55866 Related Bradford Plus Kit: 23236 | 23246 | 23200 |
| Related product: Pierce Dilution-Free BSA Protein Standards, Multichannel Pipette Compatible, 2 mg/mL | |||
Effortless, Fast, Accurate
Select Bradford assays now include Dilution-Free BSA Protein Standards which are a set of seven prediluted BSA standards, uniquely designed for multichannel pipette compatibility.
The Pierce Dilution-Free protein standards minimize tedious dilutions and pipetting errors, delivering accurate results while reducing assay setup time. They are calibrated by direct comparison to purified BSA from the National Institute of Standards and Technology (NIST) to help ensure accurate standard curve generation.
Bradford assay principles
The Bradford assay was first described by Dr. Marion Bradford in 1976 and uses Coomassie G-250 dye in a colorimetric reagent for the detection and quantitation of total protein. Pierce Bradford Plus Protein Assays are modifications of the reagent first reported by Dr. Bradford.
Chemistry of Coomassie-based Bradford protein assays
In an acidic environment, proteins bind to Coomassie dye. This results in a spectral shift from the reddish-brown form of the dye (absorbance maximum at 465 nm) to the blue form (absorbance maximum at 610 nm). The difference between the two dye forms is greatest at 595 nm, making it the optimal wavelength to measure the blue color from the Coomassie dye–protein complex. If desired, the blue color can be measured at any wavelength between 575 nm and 615 nm. At the two extremes (575 nm and 615 nm), there is an approximate 10% decrease in the measured amount of color (absorbance) compared to that obtained at 595 nm. Development of color in Coomassie dye–based protein assays has been associated with the presence of certain basic amino acids—primarily arginine, lysine, and histidine—in the protein. Van der Waals forces and hydrophobic interactions also influence dye–protein binding. The number of Coomassie dye molecules bound to each protein is approximately proportional to the number of positive charges found on the protein. Free amino acids, peptides, and low molecular weight proteins do not produce color with Coomassie dye reagents. In general, the mass of a peptide or protein should be at least 3,000 Da for quantification with this reagent. In some applications, this can be an advantage.
The main disadvantage of Bradford protein assays is their incompatibility with surfactants at concentrations routinely used to solubilize membrane proteins. In general, the presence of a surfactant in the sample, even at low concentrations, causes precipitation of the reagent. This limitation can be overcome by using Detergent Compatible Bradford Assay. In addition, the Coomassie dye reagent is highly acidic, so proteins with poor acid-solubility cannot be assayed with this reagent. Finally, Coomassie reagents result in about twice as much protein-to-protein variation as copper chelation-based assay reagents.
Product highlights
Bradford Plus Protein Assay Kit with Dilution-Free BSA Protein Standards
The Bradford Plus Protein Assay Kit with Dilution-Free BSA Protein Standards reduces assay setup by eliminating tedious standard curve generation. Dilution-Free BSA Protein Standards are available as part of the Bradford Plus Protein Assay or available separately for use with any Pierce Bradford Assay.
Figure 2. Bradford Plus Protein Assay with Dilution-Free BSA Protein Standards Assay Time.
Detergent Compatible Bradford Assay
Pierce Detergent Compatible Bradford Assay Kit is a quick and ready-to-use modification of the well-known Bradford Assay for total protein quantitation. This formulation is compatible with up to 1% of commonly used detergents. Comparing Pierce Detergent Compatible Bradford Assay to the Bio-Rad DC Protein Assay, better sensitivity is seen with the Pierce Detergent Compatible Bradford Assay using common detergents. The range of the standard curve for the Pierce Detergent Compatible Bradford assay is 4 times broader than the range for the Bio-Rad DC assay.
Figure 3. Bradford protein assay vs. the Bio-Rad DC protein assay. Each assay was performed in a microplate using BSA standards spiked with detergent or water (control), and followed the manufacturers’ instructions.
Table 1. Comparing Pierce Detergent Compatible Bradford Assay Kit with Bio-Rad DC Protein Assay Kit.
| Features | Pierce Detergent Compatible Bradford Assay Kit | Bio-Rad DC Protein Assay Kit |
|---|---|---|
| Assay measurement (absorbance maximum) | 595 nm | 750 nm |
| Test tube assay sample volume | 50 μL | 100 μL |
| Microplate assay sample volume | 10 μL | 5 μL |
| Assay working range | 100-1,500 μg/mL | 200-1,500 μg/mL |
| Absorbance range (sensitivity) | High | Low |
| Number of reagents in kit | 1 reagent | 3 reagents |
| Setup time | 10 min | 30 min |
| Incubation time | 10 min | 15 min |
| Total | 20 min | 45 min |
 | |
| Pierce 660 nm Protein Assay Kit | |
|---|---|
| Assay range: microplate (sample volume) | 25 to 2,000 µg/mL (65 µL) or 50 to 2,000 µg/mL (10 µL) |
| Incubation time and temperature | 5 |
| Total assay time | 75 |
| Absorbance | 660 nm |
| Compatible reagents | Reducing agents, chelating agents, detergents |
| Incompatible reagents | High levels of detergents or SDS requires addition of Ionic detergent compatibility reagent |
| Uniformity | Less protein–protein variation than the Coomassie (Bradford) assay |
| Product size | 450 mL/kit |
| Cat. No. | 22662 |
| Related product: Pierce Dilution-Free BSA Protein Standards, Multichannel Pipette Compatible, 2 mg/mL | |
Pierce 660 nm Assay principles
Chemistry of Pierce 660 nm Assay
The Pierce 660nm Protein Assay is based on the binding of a unique dye-metal complex to protein in acidic conditions that causes a shift in the dye's absorption maximum, which is measured at 660 nm. The dye-metal complex is reddish-brown and changes to green upon protein binding. The color change is produced by deprotonation of the dye at low pH facilitated by interactions with positively charged amino acid groups in proteins. Therefore, the dye interacts mainly with basic residues in proteins, such as histidine, arginine and lysine and to a lesser extent tyrosine, tryptophan and phenylalanine.
The color produced in the assay is stable and increases in proportion to a broad range of increasing protein concentrations, even in the presence of detergents and reducing agents that would be incompatible with Bradford and BCA Protein Assays. The optional IDCR may be added to the assay reagent to increase compatibility with high amounts of ionic detergents, allowing samples containing Laemmli SDS sample buffer with bromophenol blue to be measured. The IDCR completely dissolves by thorough mixing and does not have any effect on the assay.
Figure 4. Absorption maximum of the 660 nm Assay Reagent-metal complex shifts proportionally upon binding to BSA. Protein in the presence of the reagent-metal complex produces a significant absorbance shift at a wavelength of 660 nm.
Protein quantification with Pierce 660 nm Protein Assay
The Pierce 660 nm Assay is more linear than coomassie-based Bradford assays and compatible with higher concentrations of most detergents, reducing agents and other commonly used reagents. The accessory Ionic Detergent Compatibility Reagent (IDCR) provides for even broader detergent compatibility, making this one of the only protein assays that is suitable for samples containing Laemmli SDS sample buffer with bromophenol blue. Although the Pierce 660 nm Protein Assay produces a higher level of protein-to-protein variation (37%) than other assays, such as the BCA Protein Assay, the simpler single-reagent format and broader substance compatibility make the Pierce 660 nm Assay more convenient for many routine applications. The Pierce 660nm Protein Assay can be performed in either a test tube or microplate format.
Figure 5. Performance comparison and typical color response
A: Performance comparison of the Bio-Rad Bradford Protein Assay versus the Thermo Scientific Pierce 660nm Protein Assay. Assays were performed according the standard test-tube procedure using 100µL of BSA. The Pierce 660nm Protein Assay has a greater linear range (25 to 2000µg) compared with the Bradford Assay (125 to 1000µg). Absorbances were measured at the appropriate wavelengths for each assay (660nm and 595nm, respectively). Typical color response curve using the test tube procedure.
B: Typical color response curved using the test tube procedure. The linear detection ranges are 25 to 2000µg/mL for bovine serum albumin (BSA) and 50 to 2000µg/mL for bovine gamma globulin (BGG). Due to the inherent protein to protein variability of all protein assays (37% for the 660nm Protein Assay), this demonstrates that appropriate standards should be used for the type of unknown samples being measured.
Manuals & protocols
Application & technical guides
Other literature
| | | |
| Bradford Plus Protein Assay Kit with Dilution-Free BSA Protein Standards | Detergent Compatible Bradford Assay | Bradford Protein Assay | |
|---|---|---|---|
| Features |
|
|
|
| Assay range (sample volume) | 125–1,500 µg/mL (5 µL) Or 1–25 µg/mL (150 µL) | 100–1,500 µg/mL (10 µL) or 2–25 µg/mL (150 µL) | 100–1,500 µg/mL (20 µL) or 1–25 µg/mL (150 µL) |
| Compatible reagents | Buffer salts, metal ions, reducing agents, chelators | Detergents, buffer salts, metal ions, reducing agents, chelators | Buffer salts, metal ions, reducing agents, chelators |
| Incompatibilities | Detergents | >5% detergent solutions | Detergents |
| Incubation time | 10 min | 10 min | 10 min |
| Product size | 950 mL/kit | 450 mL/kit | 950 mL/kit |
| Cat. No. | A55866 Related Bradford Plus Kit: 23236 | 23246 | 23200 |
| Related product: Pierce Dilution-Free BSA Protein Standards, Multichannel Pipette Compatible, 2 mg/mL | |||
Effortless, Fast, Accurate
Select Bradford assays now include Dilution-Free BSA Protein Standards which are a set of seven prediluted BSA standards, uniquely designed for multichannel pipette compatibility.
The Pierce Dilution-Free protein standards minimize tedious dilutions and pipetting errors, delivering accurate results while reducing assay setup time. They are calibrated by direct comparison to purified BSA from the National Institute of Standards and Technology (NIST) to help ensure accurate standard curve generation.
Bradford assay principles
The Bradford assay was first described by Dr. Marion Bradford in 1976 and uses Coomassie G-250 dye in a colorimetric reagent for the detection and quantitation of total protein. Pierce Bradford Plus Protein Assays are modifications of the reagent first reported by Dr. Bradford.
Chemistry of Coomassie-based Bradford protein assays
In an acidic environment, proteins bind to Coomassie dye. This results in a spectral shift from the reddish-brown form of the dye (absorbance maximum at 465 nm) to the blue form (absorbance maximum at 610 nm). The difference between the two dye forms is greatest at 595 nm, making it the optimal wavelength to measure the blue color from the Coomassie dye–protein complex. If desired, the blue color can be measured at any wavelength between 575 nm and 615 nm. At the two extremes (575 nm and 615 nm), there is an approximate 10% decrease in the measured amount of color (absorbance) compared to that obtained at 595 nm. Development of color in Coomassie dye–based protein assays has been associated with the presence of certain basic amino acids—primarily arginine, lysine, and histidine—in the protein. Van der Waals forces and hydrophobic interactions also influence dye–protein binding. The number of Coomassie dye molecules bound to each protein is approximately proportional to the number of positive charges found on the protein. Free amino acids, peptides, and low molecular weight proteins do not produce color with Coomassie dye reagents. In general, the mass of a peptide or protein should be at least 3,000 Da for quantification with this reagent. In some applications, this can be an advantage.
Figure 1. Reaction schematic for the Coomassie dye–based Bradford protein assays.
The main disadvantage of Bradford protein assays is their incompatibility with surfactants at concentrations routinely used to solubilize membrane proteins. In general, the presence of a surfactant in the sample, even at low concentrations, causes precipitation of the reagent. This limitation can be overcome by using Detergent Compatible Bradford Assay. In addition, the Coomassie dye reagent is highly acidic, so proteins with poor acid-solubility cannot be assayed with this reagent. Finally, Coomassie reagents result in about twice as much protein-to-protein variation as copper chelation-based assay reagents.
Product highlights
Bradford Plus Protein Assay Kit with Dilution-Free BSA Protein Standards
The Bradford Plus Protein Assay Kit with Dilution-Free BSA Protein Standards reduces assay setup by eliminating tedious standard curve generation. Dilution-Free BSA Protein Standards are available as part of the Bradford Plus Protein Assay or available separately for use with any Pierce Bradford Assay.
Figure 2. Bradford Plus Protein Assay with Dilution-Free BSA Protein Standards Assay Time.
Detergent Compatible Bradford Assay
Pierce Detergent Compatible Bradford Assay Kit is a quick and ready-to-use modification of the well-known Bradford Assay for total protein quantitation. This formulation is compatible with up to 1% of commonly used detergents. Comparing Pierce Detergent Compatible Bradford Assay to the Bio-Rad DC Protein Assay, better sensitivity is seen with the Pierce Detergent Compatible Bradford Assay using common detergents. The range of the standard curve for the Pierce Detergent Compatible Bradford assay is 4 times broader than the range for the Bio-Rad DC assay.
Figure 3. Bradford protein assay vs. the Bio-Rad DC protein assay. Each assay was performed in a microplate using BSA standards spiked with detergent or water (control), and followed the manufacturers’ instructions.
Table 1. Comparing Pierce Detergent Compatible Bradford Assay Kit with Bio-Rad DC Protein Assay Kit.
| Features | Pierce Detergent Compatible Bradford Assay Kit | Bio-Rad DC Protein Assay Kit |
|---|---|---|
| Assay measurement (absorbance maximum) | 595 nm | 750 nm |
| Test tube assay sample volume | 50 μL | 100 μL |
| Microplate assay sample volume | 10 μL | 5 μL |
| Assay working range | 100-1,500 μg/mL | 200-1,500 μg/mL |
| Absorbance range (sensitivity) | High | Low |
| Number of reagents in kit | 1 reagent | 3 reagents |
| Setup time | 10 min | 30 min |
| Incubation time | 10 min | 15 min |
| Total | 20 min | 45 min |
| |
| Pierce 660 nm Protein Assay Kit | |
|---|---|
| Assay range: microplate (sample volume) | 25 to 2,000 µg/mL (65 µL) or 50 to 2,000 µg/mL (10 µL) |
| Incubation time and temperature | 5 |
| Total assay time | 75 |
| Absorbance | 660 nm |
| Compatible reagents | Reducing agents, chelating agents, detergents |
| Incompatible reagents | High levels of detergents or SDS requires addition of Ionic detergent compatibility reagent |
| Uniformity | Less protein–protein variation than the Coomassie (Bradford) assay |
| Product size | 450 mL/kit |
| Cat. No. | 22662 |
| Related product: Pierce Dilution-Free BSA Protein Standards, Multichannel Pipette Compatible, 2 mg/mL | |
Pierce 660 nm Assay principles
Chemistry of Pierce 660 nm Assay
The Pierce 660nm Protein Assay is based on the binding of a unique dye-metal complex to protein in acidic conditions that causes a shift in the dye's absorption maximum, which is measured at 660 nm. The dye-metal complex is reddish-brown and changes to green upon protein binding. The color change is produced by deprotonation of the dye at low pH facilitated by interactions with positively charged amino acid groups in proteins. Therefore, the dye interacts mainly with basic residues in proteins, such as histidine, arginine and lysine and to a lesser extent tyrosine, tryptophan and phenylalanine.
The color produced in the assay is stable and increases in proportion to a broad range of increasing protein concentrations, even in the presence of detergents and reducing agents that would be incompatible with Bradford and BCA Protein Assays. The optional IDCR may be added to the assay reagent to increase compatibility with high amounts of ionic detergents, allowing samples containing Laemmli SDS sample buffer with bromophenol blue to be measured. The IDCR completely dissolves by thorough mixing and does not have any effect on the assay.
Figure 4. Absorption maximum of the 660 nm Assay Reagent-metal complex shifts proportionally upon binding to BSA. Protein in the presence of the reagent-metal complex produces a significant absorbance shift at a wavelength of 660 nm.
Protein quantification with Pierce 660 nm Protein Assay
The Pierce 660 nm Assay is more linear than coomassie-based Bradford assays and compatible with higher concentrations of most detergents, reducing agents and other commonly used reagents. The accessory Ionic Detergent Compatibility Reagent (IDCR) provides for even broader detergent compatibility, making this one of the only protein assays that is suitable for samples containing Laemmli SDS sample buffer with bromophenol blue. Although the Pierce 660 nm Protein Assay produces a higher level of protein-to-protein variation (37%) than other assays, such as the BCA Protein Assay, the simpler single-reagent format and broader substance compatibility make the Pierce 660 nm Assay more convenient for many routine applications. The Pierce 660nm Protein Assay can be performed in either a test tube or microplate format.
Figure 5. Performance comparison and typical color response
A: Performance comparison of the Bio-Rad Bradford Protein Assay versus the Thermo Scientific Pierce 660nm Protein Assay. Assays were performed according the standard test-tube procedure using 100µL of BSA. The Pierce 660nm Protein Assay has a greater linear range (25 to 2000µg) compared with the Bradford Assay (125 to 1000µg). Absorbances were measured at the appropriate wavelengths for each assay (660nm and 595nm, respectively). Typical color response curve using the test tube procedure.
B: Typical color response curved using the test tube procedure. The linear detection ranges are 25 to 2000µg/mL for bovine serum albumin (BSA) and 50 to 2000µg/mL for bovine gamma globulin (BGG). Due to the inherent protein to protein variability of all protein assays (37% for the 660nm Protein Assay), this demonstrates that appropriate standards should be used for the type of unknown samples being measured.
Manuals & protocols
Application & technical guides
Other literature
Resources
Pierce Protein methods – an encyclopedia of articles about protein analysis
Protein assay technical guide – tools and reagents for improved quantitation of total or specific proteins.
Protein assay compatibility table – summarizes compatible substances for several popular protein assays.
Protein Analysis Learning Center – resources for different protein analysis techniques
Protein Biology Application Notes – journal-style articles based on research applications and proof-of-concept experiments
For Research Use Only. Not for use in diagnostic procedures.