Pierce BCA protein Assay, catalog number 23227

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.

Protein Assay Selection  Protein Assay Technical Handbook

Choose the right BCA protein assay for your sample

  Pierce Rapid Gold BCA Protein assay, catalog number A53225 Pierce BCA protein Assay, catalog number 23227 BCA Protein Assay- Reducing Agent Compatible, catalog number 23250 Pierce Micro BCA Protein Assay, catalog number 23235
  Rapid Gold BCA Protein Assay BCA Protein Assay BCA Protein Assay - Reducing Agent Compatible Micro BCA Protein Assay
Assay range: (sample volume) 20 to 2,000 µg/mL
(25 µL)
20 to 2,000 µg/mL
(25 µL)
125 to 2,000 µg/mL
(9 µL)
2 to 40 µg/mL
(150 µL) or
0.5 to 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 50 min 100 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 Coomassie dye methods Less protein:protein variation than Coomassie dye methods Significantly less (14–23%) protein:protein variation than Coomassie dye methods Less protein:protein variation than BCA, Coomassie dye, or Lowry methods
Available sizes 250 mL/kit
500 mL/kit
500 mL/kit
1 L/kit
275 mL/kit 500 mL/kit
Cat. No. A53225 23227 23250 23235

BCA kits overview

  • Rapid Gold BCA Protein Assay – green to yellow (A480 nm), two-component, precise, detergent compatible assay to measure total protein vs. protein standard in 5 minutes at room temperature
  • BCA Protein Assay – green-to-purple (A562 nm), two-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).

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).

BCA Protein assay

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.

BCA reagent- biuret 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 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 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.

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.

BCA assay standard curve, Bradford assay standard curve

Figure 3. Standard curves for protein quantitation assays. (A) Purified BSA in 0.9% saline (0–2 mg/mL) was used to generate standard curves for the Pierce Rapid Gold BCA Protein Assay and the standard BCA assay. Both assays were conducted according to the manufacturer’s protocols, in a microplate format. (B) The same method described in (A) was used to generate standard curves for the Pierce Rapid Gold BCA Protein Assay and Bio-Rad Bradford Protein Assay.

Another major advantage of the BCA protein assays is the 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. This low protein-to-protein variation leads to higher accuracy in determining protein concentration for unknown protein samples.

Protein-protein variability of BCA protein assay and Bradford protein assay

Figure 4. Accuracy of the Pierce Rapid Gold BCA Protein Assay and Bradford Protein Assay with known protein mixes. Both assays were conducted according to the respective manufacturers’ protocols, in a microplate format. Known concentrations were based on manufacturers’ indicated concentrations and confirmed by absorbance at 280 nm.

Protein quantification with Pierce BCA Protein Assay

The Pierce BCA Protein Assay produces a linear response curve (R2 > 0.95) and can be performed using two different formats based upon the dynamic range needed to detect the protein concentration of an unknown sample. The standard Pierce BCA Protein Assay detects protein concentrations from 20 to 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.

Pierce BCA Protein Assay protocol

Figure 5. BCA Protein Assay Kit protocol.

Rapid Gold BCA Protein Assay Kit protocol

This colorimetric assay provides the high sensitivity and linearity associated with the BCA assay, but in a fraction of the time it takes to perform a standard BCA assay. The assay can be completed in 5 minutes at room temperature- eliminating the need to wait or expose samples to elevated temperatures.

Pierce Rapid Gold BCA assay protocol

Figure 6. 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

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.

Reaction schematic for Pierce Modified Lowry Protein assay kit

Figure 7. Reaction schematic for the Pierce Modified Lowry Protein Assay Kit.

Pierce Modified Lowry Protein assay kit protocol

Figure 8. Pierce Modified Lowry Protein Assay protocol

Choose the right BCA protein assay for your sample

  Pierce Rapid Gold BCA Protein assay, catalog number A53225 Pierce BCA protein Assay, catalog number 23227 BCA Protein Assay- Reducing Agent Compatible, catalog number 23250 Pierce Micro BCA Protein Assay, catalog number 23235
  Rapid Gold BCA Protein Assay BCA Protein Assay BCA Protein Assay - Reducing Agent Compatible Micro BCA Protein Assay
Assay range: (sample volume) 20 to 2,000 µg/mL
(25 µL)
20 to 2,000 µg/mL
(25 µL)
125 to 2,000 µg/mL
(9 µL)
2 to 40 µg/mL
(150 µL) or
0.5 to 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 50 min 100 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 Coomassie dye methods Less protein:protein variation than Coomassie dye methods Significantly less (14–23%) protein:protein variation than Coomassie dye methods Less protein:protein variation than BCA, Coomassie dye, or Lowry methods
Available sizes 250 mL/kit
500 mL/kit
500 mL/kit
1 L/kit
275 mL/kit 500 mL/kit
Cat. No. A53225 23227 23250 23235

BCA kits overview

  • Rapid Gold BCA Protein Assay – green to yellow (A480 nm), two-component, precise, detergent compatible assay to measure total protein vs. protein standard in 5 minutes at room temperature
  • BCA Protein Assay – green-to-purple (A562 nm), two-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).

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).

BCA Protein assay

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.

BCA reagent- biuret 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 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 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.

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.

BCA assay standard curve, Bradford assay standard curve

Figure 3. Standard curves for protein quantitation assays. (A) Purified BSA in 0.9% saline (0–2 mg/mL) was used to generate standard curves for the Pierce Rapid Gold BCA Protein Assay and the standard BCA assay. Both assays were conducted according to the manufacturer’s protocols, in a microplate format. (B) The same method described in (A) was used to generate standard curves for the Pierce Rapid Gold BCA Protein Assay and Bio-Rad Bradford Protein Assay.

Another major advantage of the BCA protein assays is the 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. This low protein-to-protein variation leads to higher accuracy in determining protein concentration for unknown protein samples.

Protein-protein variability of BCA protein assay and Bradford protein assay

Figure 4. Accuracy of the Pierce Rapid Gold BCA Protein Assay and Bradford Protein Assay with known protein mixes. Both assays were conducted according to the respective manufacturers’ protocols, in a microplate format. Known concentrations were based on manufacturers’ indicated concentrations and confirmed by absorbance at 280 nm.

Protein quantification with Pierce BCA Protein Assay

The Pierce BCA Protein Assay produces a linear response curve (R2 > 0.95) and can be performed using two different formats based upon the dynamic range needed to detect the protein concentration of an unknown sample. The standard Pierce BCA Protein Assay detects protein concentrations from 20 to 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.

Pierce BCA Protein Assay protocol

Figure 5. BCA Protein Assay Kit protocol.

Rapid Gold BCA Protein Assay Kit protocol

This colorimetric assay provides the high sensitivity and linearity associated with the BCA assay, but in a fraction of the time it takes to perform a standard BCA assay. The assay can be completed in 5 minutes at room temperature- eliminating the need to wait or expose samples to elevated temperatures.

Pierce Rapid Gold BCA assay protocol

Figure 6. 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

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.

Reaction schematic for Pierce Modified Lowry Protein assay kit

Figure 7. Reaction schematic for the Pierce Modified Lowry Protein Assay Kit.

Pierce Modified Lowry Protein assay kit protocol

Figure 8. Pierce Modified Lowry Protein Assay protocol