Search Thermo Fisher Scientific
Protein Assays
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 assays | Fluorescent 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 action | Copper-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 reaction | In 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 complex | Various mechanisms of actions exist depending on the assay- but in general the assay reagent is non-fluorescent until bound to proteins |
| Advantages |
|
|
|
| Disadvantages |
|
|
|
Common protein standards for protein assays
| Bovine serum albumin (BSA) standards | Bovine gamma globulin (BGG) standards | |
|---|---|---|
| When to use |
|
|
| Available formats | Bovine 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.
| Vial | Volume of diluent | Volume and source of BSA | Final BSA concentration |
|---|---|---|---|
| A | 0 | 300 µL of stock | 2,000 µg/mL |
| B | 125 µL | 375 µL of stock | 1,500 µg/mL |
| C | 325 µL | 325 µL of stock | 1,000 µg/mL |
| D | 175 µL | 175 µL of vial B dilution | 750 µg/mL |
| E | 325 µL | 325 µL of vial C dilution | 500 µg/mL |
| F | 325 µL | 325 µL of vial E dilution | 250 µg/mL |
| G | 325 µL | 325 µL of vial F dilution | 125 µg/mL |
| H | 400 µL | 100 µL of vial G dilution | 25 µg/mL |
| I | 400 µL | 0 | 0 µL/mL = blank |
Table 2. Micro BCA Assay.
| Vial | Volume of diluent | Volume and source of BSA | Final BSA concentration |
|---|---|---|---|
| A | 0.9 mL | 0.1 mL of stock | 200 µg/mL |
| B | 0.8 mL | 0.2 mL of vial A dillution | 40 µg/mL |
| C | 0.5 mL | 0.5 mL of vial B dilution | 20 µg/mL |
| D | 0.5 mL | 0.5 mL of vial C dilution | 10 µg/mL |
| E | 0.5 mL | 0.5 mL of vial D dilution | 5 µg/mL |
| F | 0.5 mL | 0.5 mL of vial E dilution | 2.5 µg/mL |
| G | 0.5 mL | 0.4 mL of vial F dilution | 1 µg/mL |
| H | 0.5 mL | 0.5 mL of vial G dilution | 0.5 µg/mL |
| I | 1.0 mL | 0 | 0 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.
| Vial | Volume of diluent | Volume and source of BSA | Final BSA concentration |
|---|---|---|---|
| A | 0 | 300 µL of stock | 2,000 µg/mL |
| B | 125 µL | 375 µL of stock | 1,500 µg/mL |
| C | 325 µL | 325 µL of stock | 1,000 µg/mL |
| D | 175 µL | 175 µL of vial B dilution | 750 µg/mL |
| E | 325 µL | 325 µL of vial C dilution | 500 µg/mL |
| F | 325 µL | 325 µL of vial E dilution | 250 µg/mL |
| G | 325 µL | 325 µL of vial F dilution | 125 µg/mL |
| H | 400 µL | 100 µL of vial G dilution | 25 µg/mL |
| I | 400 µL | 0 | 0 µL/mL = blank |
Table 3. Micro assays.
| Vial | Volume of diluent | Volume and source of BSA | Final BSA concentration |
|---|---|---|---|
| A | 237 µL | 3 µL of stock | 25 µg/mL |
| B | 495 µL | 5 µL of stock | 20 µg/mL |
| C | 379 µL | 3 µL of stock | 15 µg/mL |
| D | 250 µL | 250 µL of vial B dilution | 10 µg/mL |
| E | 200 µL | 200 µL of vial D dilution | 5 µg/mL |
| F | 150 µL | 150 µL of vial E dilution | 2.5 µg/mL |
| G | 500 µL | 0 | 0 µL/mL = blank |
Technical handbook
| BCA and Lowry assays | Bradford and Dye assays | Fluorescent 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 action | Copper-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 reaction | In 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 complex | Various mechanisms of actions exist depending on the assay- but in general the assay reagent is non-fluorescent until bound to proteins |
| Advantages |
|
|
|
| Disadvantages |
|
|
|
Common protein standards for protein assays
| Bovine serum albumin (BSA) standards | Bovine gamma globulin (BGG) standards | |
|---|---|---|
| When to use |
|
|
| Available formats | Bovine 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.
| Vial | Volume of diluent | Volume and source of BSA | Final BSA concentration |
|---|---|---|---|
| A | 0 | 300 µL of stock | 2,000 µg/mL |
| B | 125 µL | 375 µL of stock | 1,500 µg/mL |
| C | 325 µL | 325 µL of stock | 1,000 µg/mL |
| D | 175 µL | 175 µL of vial B dilution | 750 µg/mL |
| E | 325 µL | 325 µL of vial C dilution | 500 µg/mL |
| F | 325 µL | 325 µL of vial E dilution | 250 µg/mL |
| G | 325 µL | 325 µL of vial F dilution | 125 µg/mL |
| H | 400 µL | 100 µL of vial G dilution | 25 µg/mL |
| I | 400 µL | 0 | 0 µL/mL = blank |
Table 2. Micro BCA Assay.
| Vial | Volume of diluent | Volume and source of BSA | Final BSA concentration |
|---|---|---|---|
| A | 0.9 mL | 0.1 mL of stock | 200 µg/mL |
| B | 0.8 mL | 0.2 mL of vial A dillution | 40 µg/mL |
| C | 0.5 mL | 0.5 mL of vial B dilution | 20 µg/mL |
| D | 0.5 mL | 0.5 mL of vial C dilution | 10 µg/mL |
| E | 0.5 mL | 0.5 mL of vial D dilution | 5 µg/mL |
| F | 0.5 mL | 0.5 mL of vial E dilution | 2.5 µg/mL |
| G | 0.5 mL | 0.4 mL of vial F dilution | 1 µg/mL |
| H | 0.5 mL | 0.5 mL of vial G dilution | 0.5 µg/mL |
| I | 1.0 mL | 0 | 0 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.
| Vial | Volume of diluent | Volume and source of BSA | Final BSA concentration |
|---|---|---|---|
| A | 0 | 300 µL of stock | 2,000 µg/mL |
| B | 125 µL | 375 µL of stock | 1,500 µg/mL |
| C | 325 µL | 325 µL of stock | 1,000 µg/mL |
| D | 175 µL | 175 µL of vial B dilution | 750 µg/mL |
| E | 325 µL | 325 µL of vial C dilution | 500 µg/mL |
| F | 325 µL | 325 µL of vial E dilution | 250 µg/mL |
| G | 325 µL | 325 µL of vial F dilution | 125 µg/mL |
| H | 400 µL | 100 µL of vial G dilution | 25 µg/mL |
| I | 400 µL | 0 | 0 µL/mL = blank |
Table 3. Micro assays.
| Vial | Volume of diluent | Volume and source of BSA | Final BSA concentration |
|---|---|---|---|
| A | 237 µL | 3 µL of stock | 25 µg/mL |
| B | 495 µL | 5 µL of stock | 20 µg/mL |
| C | 379 µL | 3 µL of stock | 15 µg/mL |
| D | 250 µL | 250 µL of vial B dilution | 10 µg/mL |
| E | 200 µL | 200 µL of vial D dilution | 5 µg/mL |
| F | 150 µL | 150 µL of vial E dilution | 2.5 µg/mL |
| G | 500 µL | 0 | 0 µL/mL = blank |
Technical handbook
For Research Use Only. Not for use in diagnostic procedures.