Pierce Rapid Gold BCA Protein Assay, catalog number A53226

Measuring protein concentration

Accurately quantifying total protein concentration is a key step in most experiments and workflows involving isolation, separation, and analysis of proteins by biochemical methods. The choice among available protein assays typically is based upon several factors, including its chemical compatibility with buffer components of the samples to be assayed. We offer a variety of assay reagents, kits and standards for protein quantitation by fluorescent or colorimetric detection with fluorometers, spectrophotometers, and plate readers.

 BCA and Lowry assays Bradford and Dye assaysFluorescent assays
Typical Working ranges  20–2,000 µg/mL standard protocols
0.5–40 µg/mL enhanced protocols
100–1,500 µg/mL standard protocols
1–25 µg/mL enhanced protocols
10 ng/mL to 150 µg/mL
50 µg/mL to 5 mg/mL
Mechanism of actionCopper-based protein assays, including the bicinchoninic acid (BCA) and Lowry methods, depend on the biuret reaction as a first step. In the biuret reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions (Cu2+) in an alkaline environment containing sodium potassium tartrate. Biuret reacts with copper to form a light blue tetradentate complex. Upon the addition of a second reagent, which differs between the BCA and Lowry methods, the color is enhanced, increasing the sensitivity of the biuret reactionIn the acidic environment of the reagent, protein binds to the 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 complexVarious mechanisms of actions exist depending on the assay- but in general the assay reagent is non-fluorescent until bound to proteins
Advantages
  • Compatibility with most surfactants—even if present in the sample at concentrations up to 5%
  • Linear response curve (R2 > 0.95)
  • Less protein–protein variation than the Coomassie dye–based assays
  • Fastest and easiest to perform of all protein assays 
  • Performed at room temperature
  • Compatible with most salts, solvents, buffers, thiols, reducing substances, and metal-chelating agents
  • Excellent sensitivity, requiring less protein sample for quantitation
  • Timing is not a critical factor, so the assays can be adapted for automated handling in high-throughput applications
  • Good protein-to-protein variation
Disadvantages
  • Substances that reduce copper will also produce color in the BCA assay, disrupting the accuracy of the protein quantitation
  • Reagents that chelate copper, common reducing agents such as DTT, and specific single amino acids will also produce color in the BCA assay (cysteine or cystine, tyrosine, and tryptophan
  • Time and incubation temperatures (37°C or 60°C) necessary to achieve optimal sensitivity with traditional BCA assays
  • Incompatibility with surfactants (detergents) at concentrations routinely used to solubilize membrane proteins
  • High protein–protein variation when compared to copper-based assays
  • Requires specialized equipment or fluorescent capable plate readers

Common protein standards for protein assays

 Bovine serum albumin (BSA) standardsBovine gamma globulin (BGG) standards
When to use
  • Use as standard for protein quantitation with samples not containing immunoglobulins
  • Protein recovery control for desalting and other column procedures
  • General calibration of spectrophotometer UV-lamp (absorbance at 280 nm)
  • Use as standard for quantitation of purified antibodies or immunoglobulin-rich samples (IgG)
  • Antibody recovery control for desalting and other column procedures 
  • General calibration of spectrophotometer UV-lamp (absorbance at 280 nm)
Available formatsBovine Serum Albumin Standard Pre-Diluted Set (Cat. No. 23208)
Bovine Serum Albumin Standard, 2 mg/mL, 50 mL (Cat. No. 23210)
Bovine Serum Albumin Standard Ampules, 2 mg/mL (Cat. No. 23209)
Bovine Gamma Globulin Standard Ampules, 2 mg/mL (Cat. No. 23212)
Bovine Gamma Globulin Standard Pre-Diluted Set (Cat. No. 23213)

For the greatest accuracy in estimating total protein concentration in unknown samples, it is essential to include a standard curve each time the assay is performed. This is particularly true for the protein assay methods that produce nonlinear standard curves. Determination of the number of standards and replicates used to define the standard curve depends upon the degree of nonlinearity in the standard curve and the degree of accuracy required. In general, fewer points are needed to construct a standard curve if the colorimetric response is linear. Typically, standard curves are constructed using at least two replicates for each point on the curve. Below are example standard sets that can be used for BCA and Coomassie assays.

The tables below provide information on how to prepare a set of protein standards for various Pierce protein assays. This information is only a guide that covers BSA standards; consult the information supplied with a given standard for more accurate guidelines.

Preparation of BSA standards for Pierce BCA assays and Pierce 660 nm assay standard curve

Dilution scheme using a 2 mg/ml stock.

Table 1. Standard tube or microplate protocols.

VialVolume of diluentVolume and source of BSAFinal BSA concentration
A0300 µL of stock2,000 µg/mL
B125 µL375 µL of stock1,500 µg/mL
C325 µL325 µL of stock1,000 µg/mL
D175 µL175 µL of vial B dilution750 µg/mL
E325 µL325 µL of vial C dilution500 µg/mL
F325 µL325 µL of vial E dilution250 µg/mL
G325 µL325 µL of vial F dilution125 µg/mL
H400 µL100 µL of vial G dilution25 µg/mL
I400 µL00 µL/mL = blank

Table 2. Micro BCA Assay.

VialVolume of diluentVolume and source of BSAFinal BSA concentration
A0.9 mL0.1 mL of stock200 µg/mL
B0.8 mL0.2 mL of vial A dillution40 µg/mL
C0.5 mL0.5 mL of vial B dilution20 µg/mL
D0.5 mL0.5 mL of vial C dilution10 µg/mL
E0.5 mL0.5 mL of vial D dilution5 µg/mL
F0.5 mL0.5 mL of vial E dilution2.5 µg/mL
G0.5 mL0.4 mL of vial F dilution1 µg/mL
H0.5 mL0.5 mL of vial G dilution0.5 µg/mL
I1.0 mL00 µL/mL = blank

Preparation of BSA standards for Coomassie (Bradford) assay standard curve

Dilution scheme using a 2 mg/ml stock.

Table 3. Standard tube or microplate protocols.

VialVolume of diluentVolume and source of BSAFinal BSA concentration
A0300 µL of stock2,000 µg/mL
B125 µL375 µL of stock1,500 µg/mL
C325 µL325 µL of stock1,000 µg/mL
D175 µL175 µL of vial B dilution750 µg/mL
E325 µL325 µL of vial C dilution500 µg/mL
F325 µL325 µL of vial E dilution250 µg/mL
G325 µL325 µL of vial F dilution125 µg/mL
H400 µL100 µL of vial G dilution25 µg/mL
I400 µL00 µL/mL = blank

Table 3. Micro assays.

VialVolume of diluentVolume and source of BSAFinal BSA concentration
A237 µL3 µL of stock25 µg/mL
B495 µL5 µL of stock20 µg/mL
C379 µL3 µL of stock15 µg/mL
D250 µL250 µL of vial B dilution10 µg/mL
E200 µL200 µL of vial D dilution5 µg/mL
F150 µL150 µL of vial E dilution2.5 µg/mL
G500 µL00 µL/mL = blank

Technical handbook

 BCA and Lowry assays Bradford and Dye assaysFluorescent assays
Typical Working ranges  20–2,000 µg/mL standard protocols
0.5–40 µg/mL enhanced protocols
100–1,500 µg/mL standard protocols
1–25 µg/mL enhanced protocols
10 ng/mL to 150 µg/mL
50 µg/mL to 5 mg/mL
Mechanism of actionCopper-based protein assays, including the bicinchoninic acid (BCA) and Lowry methods, depend on the biuret reaction as a first step. In the biuret reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions (Cu2+) in an alkaline environment containing sodium potassium tartrate. Biuret reacts with copper to form a light blue tetradentate complex. Upon the addition of a second reagent, which differs between the BCA and Lowry methods, the color is enhanced, increasing the sensitivity of the biuret reactionIn the acidic environment of the reagent, protein binds to the 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 complexVarious mechanisms of actions exist depending on the assay- but in general the assay reagent is non-fluorescent until bound to proteins
Advantages
  • Compatibility with most surfactants—even if present in the sample at concentrations up to 5%
  • Linear response curve (R2 > 0.95)
  • Less protein–protein variation than the Coomassie dye–based assays
  • Fastest and easiest to perform of all protein assays 
  • Performed at room temperature
  • Compatible with most salts, solvents, buffers, thiols, reducing substances, and metal-chelating agents
  • Excellent sensitivity, requiring less protein sample for quantitation
  • Timing is not a critical factor, so the assays can be adapted for automated handling in high-throughput applications
  • Good protein-to-protein variation
Disadvantages
  • Substances that reduce copper will also produce color in the BCA assay, disrupting the accuracy of the protein quantitation
  • Reagents that chelate copper, common reducing agents such as DTT, and specific single amino acids will also produce color in the BCA assay (cysteine or cystine, tyrosine, and tryptophan
  • Time and incubation temperatures (37°C or 60°C) necessary to achieve optimal sensitivity with traditional BCA assays
  • Incompatibility with surfactants (detergents) at concentrations routinely used to solubilize membrane proteins
  • High protein–protein variation when compared to copper-based assays
  • Requires specialized equipment or fluorescent capable plate readers

Common protein standards for protein assays

 Bovine serum albumin (BSA) standardsBovine gamma globulin (BGG) standards
When to use
  • Use as standard for protein quantitation with samples not containing immunoglobulins
  • Protein recovery control for desalting and other column procedures
  • General calibration of spectrophotometer UV-lamp (absorbance at 280 nm)
  • Use as standard for quantitation of purified antibodies or immunoglobulin-rich samples (IgG)
  • Antibody recovery control for desalting and other column procedures 
  • General calibration of spectrophotometer UV-lamp (absorbance at 280 nm)
Available formatsBovine Serum Albumin Standard Pre-Diluted Set (Cat. No. 23208)
Bovine Serum Albumin Standard, 2 mg/mL, 50 mL (Cat. No. 23210)
Bovine Serum Albumin Standard Ampules, 2 mg/mL (Cat. No. 23209)
Bovine Gamma Globulin Standard Ampules, 2 mg/mL (Cat. No. 23212)
Bovine Gamma Globulin Standard Pre-Diluted Set (Cat. No. 23213)

For the greatest accuracy in estimating total protein concentration in unknown samples, it is essential to include a standard curve each time the assay is performed. This is particularly true for the protein assay methods that produce nonlinear standard curves. Determination of the number of standards and replicates used to define the standard curve depends upon the degree of nonlinearity in the standard curve and the degree of accuracy required. In general, fewer points are needed to construct a standard curve if the colorimetric response is linear. Typically, standard curves are constructed using at least two replicates for each point on the curve. Below are example standard sets that can be used for BCA and Coomassie assays.

The tables below provide information on how to prepare a set of protein standards for various Pierce protein assays. This information is only a guide that covers BSA standards; consult the information supplied with a given standard for more accurate guidelines.

Preparation of BSA standards for Pierce BCA assays and Pierce 660 nm assay standard curve

Dilution scheme using a 2 mg/ml stock.

Table 1. Standard tube or microplate protocols.

VialVolume of diluentVolume and source of BSAFinal BSA concentration
A0300 µL of stock2,000 µg/mL
B125 µL375 µL of stock1,500 µg/mL
C325 µL325 µL of stock1,000 µg/mL
D175 µL175 µL of vial B dilution750 µg/mL
E325 µL325 µL of vial C dilution500 µg/mL
F325 µL325 µL of vial E dilution250 µg/mL
G325 µL325 µL of vial F dilution125 µg/mL
H400 µL100 µL of vial G dilution25 µg/mL
I400 µL00 µL/mL = blank

Table 2. Micro BCA Assay.

VialVolume of diluentVolume and source of BSAFinal BSA concentration
A0.9 mL0.1 mL of stock200 µg/mL
B0.8 mL0.2 mL of vial A dillution40 µg/mL
C0.5 mL0.5 mL of vial B dilution20 µg/mL
D0.5 mL0.5 mL of vial C dilution10 µg/mL
E0.5 mL0.5 mL of vial D dilution5 µg/mL
F0.5 mL0.5 mL of vial E dilution2.5 µg/mL
G0.5 mL0.4 mL of vial F dilution1 µg/mL
H0.5 mL0.5 mL of vial G dilution0.5 µg/mL
I1.0 mL00 µL/mL = blank

Preparation of BSA standards for Coomassie (Bradford) assay standard curve

Dilution scheme using a 2 mg/ml stock.

Table 3. Standard tube or microplate protocols.

VialVolume of diluentVolume and source of BSAFinal BSA concentration
A0300 µL of stock2,000 µg/mL
B125 µL375 µL of stock1,500 µg/mL
C325 µL325 µL of stock1,000 µg/mL
D175 µL175 µL of vial B dilution750 µg/mL
E325 µL325 µL of vial C dilution500 µg/mL
F325 µL325 µL of vial E dilution250 µg/mL
G325 µL325 µL of vial F dilution125 µg/mL
H400 µL100 µL of vial G dilution25 µg/mL
I400 µL00 µL/mL = blank

Table 3. Micro assays.

VialVolume of diluentVolume and source of BSAFinal BSA concentration
A237 µL3 µL of stock25 µg/mL
B495 µL5 µL of stock20 µg/mL
C379 µL3 µL of stock15 µg/mL
D250 µL250 µL of vial B dilution10 µg/mL
E200 µL200 µL of vial D dilution5 µg/mL
F150 µL150 µL of vial E dilution2.5 µg/mL
G500 µL00 µL/mL = blank

Technical handbook