Copper-based total protein quantification
The BCA Protein Assay is a popular method for colorimetric detection and quantitation of total protein. Pierce BCA Protein Assays have a unique advantage over the Coomassie dye–based assays (Bradford)–they are compatible with samples that contain up to 5% surfactants (detergents), and are affected much less by protein compositional differences, providing greater protein-to-protein uniformity.
For added ease-of-use, BCA protein assays with Dilution-Free protein standards reduce assay setup time by up to 80%. Dilution-Free protein standards are a set of prediluted standards packaged in a multichannel pipette-compatible format that enable you to easily transfer a full dilution series to your microplate at once.
Choose the right BCA protein assay for your sample
 |  |  |  | |
| Pierce Dilution-Free Rapid Gold BCA Protein Assay | Pierce BCA Protein Assay Kit with Dilution-Free BSA Protein Standards | Pierce BCA Protein Assay - Reducing Agent Compatible | Micro BCA Protein Assay | |
|---|---|---|---|---|
| Assay range: (sample volume) | 20–10,000 µg (10 µL) | 20–2,000 µg/mL (25 µL) | 125–2,000 µg/mL (9 µL) | 2–40 µg/mL (150 µL) or 0.5–20 µg/mL (500 µL) |
| Incubation time and temperature | 5 min at RT | 30 min at 37°C | 45 min at 37°C | 60 min at 60°C |
| Total assay time | 10 min | 45 min | 115 min | 130 min |
| Absorbance | 480 nm | 562 nm | 562 nm | 562 nm |
| Compatible reagents | Detergents | Detergents | Detergents/reducing agents (e.g. DTT) | Detergents |
| Incompatible reagents | Reducing agents; chelators | Reducing agents; chelators | Chelators | Reducing agents; chelators |
| Uniformity | Less protein: protein variation than Bradford assays | Less protein: protein variation than Bradford assays | Significantly less (14–23%) protein: protein variation than Bradford assays | Less protein: protein variation than Bradford assays |
| Dilution-Free protein standards | Yes | Yes | No | No |
| Cat. No. | A55860 (500 mL) A55861 (250 mL) A55862 (20 mL) | A55864 (1000 mL) A55865 (500 mL) Related BCA Kits: 23225 (1000 mL) A65453 (100 mL) 23227 (500 mL) | 23250 (275 mL/kit) 23252 (microplate) | 23235 (500 mL/kit) |
| Related product: Pierce Dilution-Free BSA Protein Standards, Multichannel Pipette Compatible, 0.125–2 mg/mL Related product: Pierce Dilution-Free BSA Protein Standards, Multichannel Pipette Compatible, 0.125–10 mg/mL | ||||
BCA kits overview
- Dilution-Free Rapid Gold BCA Protein Assay – green to yellow (A480 nm), 2-component, precise, detergent compatible assay to measure total protein vs. protein standard in 5 minutes at room temperature
- BCA Protein Assay with Dilution-Free BSA Protein Standards – green-to-purple (A562 nm), 2-component, precise, detergent-compatible assay reagent to measure total protein concentration vs. protein standard in 30 minutes at 37°C.
- BCA Protein Assay - Reducing Agent Compatible – reducing agent–compatible version of our BCA Reagent Kit to measure protein concentration in samples containing DTT or BME thiol-reductants.
- Micro BCA Protein Assay – special 3-component version of our BCA Reagent Kit to measure total protein concentration of dilute protein solutions (0.5 to 20 μg/mL).
Effortless, Fast, Accurate
Select BCA assays now include Dilution-Free BSA Protein Standards which are a set of 7 prediluted BSA standards, uniquely designed for multichannel pipette compatibility.
Pierce Dilution-Free standards allow you to eliminate time-consuming and tedious dilutions when generating your standard curve and are calibrated by direct comparison to purified BSA from the National Institute of Standards and Technology (NIST) to help ensure accurate standard curve generation.
BCA assay principles
Chemistry of BCA-based protein assays
The Pierce BCA Protein Assays combines the well-known reduction of Cu2+ to Cu+ by protein in an alkaline medium (also known as the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) by bicinchoninic acid (BCA).
Figure 1. The first step is the chelation of copper with protein in an alkaline environment to form a blue-colored complex. In this reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate.
Single amino acids and dipeptides are unaffected in the biuret reaction, but tripeptides and larger polypeptides or proteins will react to produce the light blue-to-violet complex that absorbs light at 540 nm. One cupric ion forms a colored coordination complex with four to six nearby peptides bonds. The intensity of the color produced is proportional to the number of peptide bonds participating in the reaction.
Figure 2. In the second step, BCA reagent, a highly sensitive and selective colorimetric detection reagent, reacts with the cuprous cation (Cu+) that was formed in the first step and produces a purple-color.
The BCA-copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The purple color may be measured at any wavelength between 550 and 570 nm with minimal (less than 10%) loss of signal. The signal induced by the BCA reagent is approximately 100 times more sensitive (lower limit of detection) than the signal using the biuret reagent.
Pierce Dilution-Free Rapid Gold BCA protein assay uses the same copper reduction method as traditional BCA assay with a unique copper chelator. In the second step of the color development reaction, the Pierce Dilution-Free Rapid Gold BCA chelator reacts with the reduced (cuprous) cation (Cu+) that was formed in step one to produce an intensely gold-colored reaction product. The copper complex exhibits a strong linear absorbance at 480 nm with increasing protein concentrations.
Dilution-Free BCA protein assays
Dilution-Free BSA Protein Standards are available separately or as part of select protein assay kits. Compared to the Bradford Assay or Pierce BCA Protein Assay Kit, the Dilution-Free Rapid Gold BCA Protein Assay reduces assay setup time by up to 80% by eliminating the need to dilute samples and standards. Also, it requires only a 5-minute room temperature incubation. The Dilution-Free BSA Protein Standards are also available in the BCA Protein Assay Kit with Dilution-Free BSA Protein Standards.
Figure 3. The Pierce Dilution-Free Rapid Gold BCA Assay reduces setup time by up to 80% compared to other protein assays. The assays were conducted following the manufacturer’s protocols in a microplate format. Five cell lysates and five pure proteins were prepared to give a total of ten samples. Standard curves for the BCA and Bradford assays were generated through serial dilution of 2 mg/mL bovine serum albumin (BSA) standard. The standard curve for the Pierce Dilution-Free Rapid Gold BCA Assay was generated using Pierce Dilution-Free BSA Protein Standards (included with the kit) packaged in a multichannel pipette-friendly tube strip. Four of the ten samples were expected to have a starting concentration of >2 mg/mL, which necessitated a sample dilution step for the BCA and Bradford assays, whereas samples were not required to be diluted for the Dilution-Free Rapid Gold BCA Assay. Samples were then mixed with working reagent from each assay and incubated according to the manufacturer’s instructions.
Advantages of BCA assays
BCA protein quantification
One of the major advantages of the BCA assay is that it produces a linear response curve. This response curve allows accurate determination of unknown protein concentrations and provides a higher dynamic range than other standard assays.
Figure 4. Pierce Dilution-Free Rapid Gold BCA Protein Assay generates a linear standard curve across a wide dynamic range (0–10 mg/mL). Standard curves were produced for the Pierce Dilution-Free Rapid Gold BCA Protein Assay and the Quick Start Bradford Protein Assay (Bio-Rad) with purified BSA in 0.9% saline (0–10 mg/mL). Both assays were conducted using the manufacturer’s protocols in a microplate format. Absorbance readings were taken using the Thermo Scientific Varioskan LUX Multimode Microplate Reader.
BCA assay accuracy
Another major advantage of BCA protein assays is the accuracy as shown through low protein-to-protein variation. Proteins are diverse in their composition and structure, and with some assays, the proteins’ differences in amino acid sequence, isoelectric point (pI), secondary structure, and side chains or prosthetic groups result in variation in the colorimetric response. Since BCA chemistries develop a color response in proportion to the number of peptide bonds present, they are less affected by these differences when compared to dye-binding assays (e.g. Bradford Assays) and therefore exhibit less protein-to-protein variation.
Figure 5. Protein quantification results of the Pierce Dilution-Free Rapid Gold BCA Protein Assay and the Quick Start Bradford Protein Assay compared to samples of known concentration. Both assays were conducted in a microplate format, according to the respective manufacturers’ protocols. For the Bradford assay, 5 μL of sample was added to 250 μL of the Bradford working reagent and incubated at room temperature for 5 minutes. For the Dilution-Free Rapid Gold BCA Protein Assay, 10 μL of sample was added to 200 μL of Dilution-Free Rapid Gold BCA working reagent and incubated at room temperature for 5 minutes. Known concentrations of protein samples were based on manufacturers’ indicated concentrations and were confirmed by absorbance at 280 nm.
BCA protein assay protocols
BCA Protein Assay with Dilution-Free BSA Protein Standards
The Pierce BCA Protein Assay can be performed using two different formats based upon the dynamic range needed to detect the protein concentration of an unknown sample. This BCA assay detects protein concentrations from 20–2,000 μg/mL using a two-component system: Reagent A, a carbonate buffer containing BCA reagent, and Reagent B, a cupric sulfate solution, which are combined to make an apple green–colored working solution that turns purple after 30 minutes at 37°C in the presence of protein. Since the color reaction is not a true endpoint reaction, considerable protocol flexibility is allowed with the Pierce BCA Protein Assay. By increasing the incubation temperature, the sensitivity of the assay can be increased. When using the enhanced tube protocol (i.e., incubating at 60°C for 30 minutes), the working range for the assay shifts to 5–250 μg/mL, facilitating detection of more dilute samples.
Figure 6. BCA Protein Assay Kit with Dilution-Free BSA Protein Standards, multichannel pipe compatible protocol.
Dilution-Free Rapid Gold BCA Protein Assay Kit protocol
The Dilution-Free Rapid Gold BCA Protein Assay provides the high sensitivity and linearity associated with the BCA assay but performed within a fraction of the time. Simply mix the BCA working solution, add samples and dilution-free standards to your microplate, incubate for 5 minutes at room temperature, and read the absorbance.
Figure 7. Dilution-Free Rapid Gold BCA Protein Assay Kit protocol.
 Modified Lowry Protein Assay Kit | |
| Assay range: microplate (sample volume) | 10 to 1,500 µg/mL (40 µL) |
| Assay range: cuvette (sample volume) | 1–1,500 µg/mL (200 µL) |
| Incubation time and temperature | 10 and 30 min at RT |
| Total assay time | 110 min |
| Absorbance | 750 nm |
| Compatible reagents | SDS (up to 1%) |
| Incompatible reagents | Reducing reagents, Chelators, detergents, tris, tricine, glycerol |
| Uniformity | Less protein:protein variation than Coomassie dye methods |
| Product size | 530 mL/kit |
| Cat. No. | 23240 |
| Related product: Pierce Dilution Free BSA Protein Standards, Multichannel Pipette Compatible, 2 mg/mL | |
Lowry assay principles
The Pierce Modified Lowry Protein Assay is based on the colorimetric method introduced by Oliver H. Lowry in 1951. The Pierce modified assay replaces two of the assay’s traditional unstable reagents with a single, more stable one.
Chemistry of the Pierce Modified Lowry Assay
The Pierce Modified Lowry Protein Assay is an enhanced biuret assay involving copper-chelation chemistry. Although the mechanism of color formation for the Pierce Modified Lowry Protein Assay is similar to that of the Pierce BCA Protein Assay, there are several significant differences between the two. While the precise mechanism for color formation using the Pierce Modified Lowry Protein Assay is not completely understood, it is known that the reaction occurs as two distinct steps. First, protein reacts with alkaline cupric sulfate in the presence of tartrate during a 10-minute incubation at room temperature. During this incubation, a tetradentate copper complex forms from four peptide bonds and one atom of copper, which is light blue in color (this is the “biuret reaction”). Following the incubation, the Folin phenol reagent is added. It is believed that the color enhancement occurs when the tetradentate copper complex transfers electrons to the phosphomolybdic–phosphotungstic acid complex (the Folin phenol reagent). The reduced phosphomolybdic–phosphotungstic acid complex produced by this reaction is intensely blue in color.
Figure 8. Reaction schematic for the Pierce Modified Lowry Protein Assay Kit.
Figure 9. Pierce Modified Lowry Protein Assay protocol
Manuals & protocols
Application & technical guides
Other literature
| | | | |
| Pierce Dilution-Free Rapid Gold BCA Protein Assay | Pierce BCA Protein Assay Kit with Dilution-Free BSA Protein Standards | Pierce BCA Protein Assay - Reducing Agent Compatible | Micro BCA Protein Assay | |
|---|---|---|---|---|
| Assay range: (sample volume) | 20–10,000 µg (10 µL) | 20–2,000 µg/mL (25 µL) | 125–2,000 µg/mL (9 µL) | 2–40 µg/mL (150 µL) or 0.5–20 µg/mL (500 µL) |
| Incubation time and temperature | 5 min at RT | 30 min at 37°C | 45 min at 37°C | 60 min at 60°C |
| Total assay time | 10 min | 45 min | 115 min | 130 min |
| Absorbance | 480 nm | 562 nm | 562 nm | 562 nm |
| Compatible reagents | Detergents | Detergents | Detergents/reducing agents (e.g. DTT) | Detergents |
| Incompatible reagents | Reducing agents; chelators | Reducing agents; chelators | Chelators | Reducing agents; chelators |
| Uniformity | Less protein: protein variation than Bradford assays | Less protein: protein variation than Bradford assays | Significantly less (14–23%) protein: protein variation than Bradford assays | Less protein: protein variation than Bradford assays |
| Dilution-Free protein standards | Yes | Yes | No | No |
| Cat. No. | A55860 (500 mL) A55861 (250 mL) A55862 (20 mL) | A55864 (1000 mL) A55865 (500 mL) Related BCA Kits: 23225 (1000 mL) A65453 (100 mL) 23227 (500 mL) | 23250 (275 mL/kit) 23252 (microplate) | 23235 (500 mL/kit) |
| Related product: Pierce Dilution-Free BSA Protein Standards, Multichannel Pipette Compatible, 0.125–2 mg/mL Related product: Pierce Dilution-Free BSA Protein Standards, Multichannel Pipette Compatible, 0.125–10 mg/mL | ||||
BCA kits overview
- Dilution-Free Rapid Gold BCA Protein Assay – green to yellow (A480 nm), 2-component, precise, detergent compatible assay to measure total protein vs. protein standard in 5 minutes at room temperature
- BCA Protein Assay with Dilution-Free BSA Protein Standards – green-to-purple (A562 nm), 2-component, precise, detergent-compatible assay reagent to measure total protein concentration vs. protein standard in 30 minutes at 37°C.
- BCA Protein Assay - Reducing Agent Compatible – reducing agent–compatible version of our BCA Reagent Kit to measure protein concentration in samples containing DTT or BME thiol-reductants.
- Micro BCA Protein Assay – special 3-component version of our BCA Reagent Kit to measure total protein concentration of dilute protein solutions (0.5 to 20 μg/mL).
Effortless, Fast, Accurate
Select BCA assays now include Dilution-Free BSA Protein Standards which are a set of 7 prediluted BSA standards, uniquely designed for multichannel pipette compatibility.
Pierce Dilution-Free standards allow you to eliminate time-consuming and tedious dilutions when generating your standard curve and are calibrated by direct comparison to purified BSA from the National Institute of Standards and Technology (NIST) to help ensure accurate standard curve generation.
BCA assay principles
Chemistry of BCA-based protein assays
The Pierce BCA Protein Assays combines the well-known reduction of Cu2+ to Cu+ by protein in an alkaline medium (also known as the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) by bicinchoninic acid (BCA).
Figure 1. The first step is the chelation of copper with protein in an alkaline environment to form a blue-colored complex. In this reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate.
Single amino acids and dipeptides are unaffected in the biuret reaction, but tripeptides and larger polypeptides or proteins will react to produce the light blue-to-violet complex that absorbs light at 540 nm. One cupric ion forms a colored coordination complex with four to six nearby peptides bonds. The intensity of the color produced is proportional to the number of peptide bonds participating in the reaction.
Figure 2. In the second step, BCA reagent, a highly sensitive and selective colorimetric detection reagent, reacts with the cuprous cation (Cu+) that was formed in the first step and produces a purple-color.
The BCA-copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The purple color may be measured at any wavelength between 550 and 570 nm with minimal (less than 10%) loss of signal. The signal induced by the BCA reagent is approximately 100 times more sensitive (lower limit of detection) than the signal using the biuret reagent.
Pierce Dilution-Free Rapid Gold BCA protein assay uses the same copper reduction method as traditional BCA assay with a unique copper chelator. In the second step of the color development reaction, the Pierce Dilution-Free Rapid Gold BCA chelator reacts with the reduced (cuprous) cation (Cu+) that was formed in step one to produce an intensely gold-colored reaction product. The copper complex exhibits a strong linear absorbance at 480 nm with increasing protein concentrations.
Dilution-Free BCA protein assays
Dilution-Free BSA Protein Standards are available separately or as part of select protein assay kits. Compared to the Bradford Assay or Pierce BCA Protein Assay Kit, the Dilution-Free Rapid Gold BCA Protein Assay reduces assay setup time by up to 80% by eliminating the need to dilute samples and standards. Also, it requires only a 5-minute room temperature incubation. The Dilution-Free BSA Protein Standards are also available in the BCA Protein Assay Kit with Dilution-Free BSA Protein Standards.
Figure 3. The Pierce Dilution-Free Rapid Gold BCA Assay reduces setup time by up to 80% compared to other protein assays. The assays were conducted following the manufacturer’s protocols in a microplate format. Five cell lysates and five pure proteins were prepared to give a total of ten samples. Standard curves for the BCA and Bradford assays were generated through serial dilution of 2 mg/mL bovine serum albumin (BSA) standard. The standard curve for the Pierce Dilution-Free Rapid Gold BCA Assay was generated using Pierce Dilution-Free BSA Protein Standards (included with the kit) packaged in a multichannel pipette-friendly tube strip. Four of the ten samples were expected to have a starting concentration of >2 mg/mL, which necessitated a sample dilution step for the BCA and Bradford assays, whereas samples were not required to be diluted for the Dilution-Free Rapid Gold BCA Assay. Samples were then mixed with working reagent from each assay and incubated according to the manufacturer’s instructions.
Advantages of BCA assays
BCA protein quantification
One of the major advantages of the BCA assay is that it produces a linear response curve. This response curve allows accurate determination of unknown protein concentrations and provides a higher dynamic range than other standard assays.
Figure 4. Pierce Dilution-Free Rapid Gold BCA Protein Assay generates a linear standard curve across a wide dynamic range (0–10 mg/mL). Standard curves were produced for the Pierce Dilution-Free Rapid Gold BCA Protein Assay and the Quick Start Bradford Protein Assay (Bio-Rad) with purified BSA in 0.9% saline (0–10 mg/mL). Both assays were conducted using the manufacturer’s protocols in a microplate format. Absorbance readings were taken using the Thermo Scientific Varioskan LUX Multimode Microplate Reader.
BCA assay accuracy
Another major advantage of BCA protein assays is the accuracy as shown through low protein-to-protein variation. Proteins are diverse in their composition and structure, and with some assays, the proteins’ differences in amino acid sequence, isoelectric point (pI), secondary structure, and side chains or prosthetic groups result in variation in the colorimetric response. Since BCA chemistries develop a color response in proportion to the number of peptide bonds present, they are less affected by these differences when compared to dye-binding assays (e.g. Bradford Assays) and therefore exhibit less protein-to-protein variation.
Figure 5. Protein quantification results of the Pierce Dilution-Free Rapid Gold BCA Protein Assay and the Quick Start Bradford Protein Assay compared to samples of known concentration. Both assays were conducted in a microplate format, according to the respective manufacturers’ protocols. For the Bradford assay, 5 μL of sample was added to 250 μL of the Bradford working reagent and incubated at room temperature for 5 minutes. For the Dilution-Free Rapid Gold BCA Protein Assay, 10 μL of sample was added to 200 μL of Dilution-Free Rapid Gold BCA working reagent and incubated at room temperature for 5 minutes. Known concentrations of protein samples were based on manufacturers’ indicated concentrations and were confirmed by absorbance at 280 nm.
BCA protein assay protocols
BCA Protein Assay with Dilution-Free BSA Protein Standards
The Pierce BCA Protein Assay can be performed using two different formats based upon the dynamic range needed to detect the protein concentration of an unknown sample. This BCA assay detects protein concentrations from 20–2,000 μg/mL using a two-component system: Reagent A, a carbonate buffer containing BCA reagent, and Reagent B, a cupric sulfate solution, which are combined to make an apple green–colored working solution that turns purple after 30 minutes at 37°C in the presence of protein. Since the color reaction is not a true endpoint reaction, considerable protocol flexibility is allowed with the Pierce BCA Protein Assay. By increasing the incubation temperature, the sensitivity of the assay can be increased. When using the enhanced tube protocol (i.e., incubating at 60°C for 30 minutes), the working range for the assay shifts to 5–250 μg/mL, facilitating detection of more dilute samples.
Figure 6. BCA Protein Assay Kit with Dilution-Free BSA Protein Standards, multichannel pipe compatible protocol.
Dilution-Free Rapid Gold BCA Protein Assay Kit protocol
The Dilution-Free Rapid Gold BCA Protein Assay provides the high sensitivity and linearity associated with the BCA assay but performed within a fraction of the time. Simply mix the BCA working solution, add samples and dilution-free standards to your microplate, incubate for 5 minutes at room temperature, and read the absorbance.
Figure 7. Dilution-Free Rapid Gold BCA Protein Assay Kit protocol.
Modified Lowry Protein Assay Kit | |
| Assay range: microplate (sample volume) | 10 to 1,500 µg/mL (40 µL) |
| Assay range: cuvette (sample volume) | 1–1,500 µg/mL (200 µL) |
| Incubation time and temperature | 10 and 30 min at RT |
| Total assay time | 110 min |
| Absorbance | 750 nm |
| Compatible reagents | SDS (up to 1%) |
| Incompatible reagents | Reducing reagents, Chelators, detergents, tris, tricine, glycerol |
| Uniformity | Less protein:protein variation than Coomassie dye methods |
| Product size | 530 mL/kit |
| Cat. No. | 23240 |
| Related product: Pierce Dilution Free BSA Protein Standards, Multichannel Pipette Compatible, 2 mg/mL | |
Lowry assay principles
The Pierce Modified Lowry Protein Assay is based on the colorimetric method introduced by Oliver H. Lowry in 1951. The Pierce modified assay replaces two of the assay’s traditional unstable reagents with a single, more stable one.
Chemistry of the Pierce Modified Lowry Assay
The Pierce Modified Lowry Protein Assay is an enhanced biuret assay involving copper-chelation chemistry. Although the mechanism of color formation for the Pierce Modified Lowry Protein Assay is similar to that of the Pierce BCA Protein Assay, there are several significant differences between the two. While the precise mechanism for color formation using the Pierce Modified Lowry Protein Assay is not completely understood, it is known that the reaction occurs as two distinct steps. First, protein reacts with alkaline cupric sulfate in the presence of tartrate during a 10-minute incubation at room temperature. During this incubation, a tetradentate copper complex forms from four peptide bonds and one atom of copper, which is light blue in color (this is the “biuret reaction”). Following the incubation, the Folin phenol reagent is added. It is believed that the color enhancement occurs when the tetradentate copper complex transfers electrons to the phosphomolybdic–phosphotungstic acid complex (the Folin phenol reagent). The reduced phosphomolybdic–phosphotungstic acid complex produced by this reaction is intensely blue in color.
Figure 8. Reaction schematic for the Pierce Modified Lowry Protein Assay Kit.
Figure 9. Pierce Modified Lowry Protein Assay protocol
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.