|
 Protein quantitation is often necessary before processing protein samples for isolation, separation and analysis by chromatographic, electrophoretic and immunochemical methods. Most colorimetric protein assay methods can be divided into two groups: those involving protein-copper chelation with secondary detection of the reduced copper and those based on protein-dye binding with direct detection of the color change associated with the bound dye.
The Thermo Scientific Pierce BCA and Modified Lowry Protein Assays are based on copper chelation. The Thermo Scientific Pierce 660 nm and Coomassie (Bradford) Protein Assays are based on dye binding. All are well-characterized, robust assays that provide consistent, reliable results. Collectively, they represent the state-of-the-art for colorimetric detection and quantitation of total protein.
Page Contents:
 |
Learn more...
Overview of Protein Assays
Chemistry of Protein Assays
Related literature...
Protein Assay Technical Handbook
Protein Assay Poster
|
Choosing a Protein Assay and a Protein Standard
|
Selecting a Protein Assay
As convenient and helpful as it would be, no protein assay method exists that is both perfectly specific to proteins (i.e., not affected by any nonprotein components) or uniformly sensitive to all protein types (i.e., not affected by differences in protein composition). Therefore, successful use of protein assays involves selecting an assay method that is most compatible with the samples to be analyzed, choosing an appropriate assay standard, and understanding and controlling the particular assumptions and limitations that remain.
When it is necessary to determine total protein concentration in a sample, one must first select an appropriate protein assay method. The choice among available protein assays is usually based upon compatibility of the method with the samples to be assayed. The objective is to select a method that requires the least manipulation or pretreatment of the samples containing substances that may interfere with the assay. Each method has its advantages and disadvantages. Because no one reagent can be considered the ideal or best protein assay method for all circumstances, most researchers have more than one type of protein assay available in their laboratories. The Pierce BCA Protein Assay and Coomassie (Bradford) Protein Assay complement one another and provide the two basic methods for accommodating most samples. The various accessory reagents and alternative versions of these two assays accommodate many other particular sample needs.
|
Thermo Scientific Pierce
Protein Assays...
BCA
BCA-Reducing Agent Compatible
Micro BCA
660 nm
Coomassie Plus (Bradford)
Coomassie (Bradford)
Modified Lowry
Download documents...
Tech Tip #68: Protein assay compatibility table
|
Selecting a Protein Standard
The best choice for a reference standard is a purified, known concentration of the predominant protein found in the samples. This is not always possible or necessary; in many cases, all that is needed is an estimate of the total protein concentration in the sample. If a highly purified version of the protein of interest is not available or it is too expensive to use as the standard, the alternative is to choose a protein that will produce a very similar color response curve with the selected protein assay method and is readily available to any laboratory at any time. For general protein assay work, bovine serum albumin (BSA) works well for a protein standard because it is widely available in high purity and relatively inexpensive. Although it is a mixture containing several immunoglobulins, bovine gamma globulin (BGG) also is a good standard when determining the concentration of antibodies because BGG produces a color response curve that is very similar to that of immunoglobulin G (IgG).
|
Pierce Protein Assay Standards...
BSA Protein Assay Standards
BGG Protein Assay Standards
|
Table of Protein Assay Specifications and Options
|
Guide to Thermo Scientific Protein Assay Kits and Reagents. Assay Ranges (columns 3 and 4) are expressed in micrograms per milliliter (µg/mL), followed by required sample volume is in parentheses. For Cuvettes, the values are for 1mL total assay volume with measurement in 1cm-diameter cuvette. For Microplates, the values are for 200 to 300µL total assay volume with measurement in 96-well microplate. Certain assays have two protocols: broad range and low range; both ranges are specified.
Pierce Protein
Assay Product |
Detection
(Amax) |
Cuvette
Assay Range |
Microplate
Assay Range |
Compatibilities
(Note 1) |
Uniformity
(Note 3) |
| BCA |
562nm |
20 to 2000 (50µL)
5 to 250 (50µL) |
20 to 2000 (25µL) |
Yes: Detergents
No: Reducing agents; Chelators |
High |
BCA - Reducing
Agent Compatible |
562nm |
125 to 2000 (25µL) |
125 to 2000 (9µL) |
Yes: Detergents; Reducing agents
No: Chelators |
High |
| Micro BCA |
562nm |
0.5 to 20 (500µL) |
2 to 40 (150µL) |
Yes: Detergents
No: Reducing agents; Chelators |
High |
| 660 nm |
660nm |
25 to 2000 (65µL) |
50 to 2000 (10µL) |
Yes: Detergents; Reducing agents; SDS sample buffer (Note 2)
No: Ionic detergents (unless IDCR is used, Note 2) |
Low |
| Coomassie Plus |
595nm |
100 to 1500 (35µL)
1 to 25 (500µL) |
100 to 1500 (7µL)
1 to 25 (150µL) |
Yes: Most reducing agents; Chelators
No: Detergents |
Medium |
| Coomassie (Bradford) |
595nm |
100 to 1500 (20µL)
1 to 25 (500µL) |
100 to 1500 (5µL)
1 to 25 (150µL) |
Yes: Most reducing agents; Chelators
No: Detergents |
Low |
| Modified Lowry |
750nm |
10 to 1500 (200µL) |
10 to 1500 (40µL) |
Yes: SDS
No: Most detergents; Reducing agents; Chelators |
High |
Notes:
1. Levels typically used in cell lysis reagents and protein buffers.
2. Possible when the Ionic Detergent Compatibility Reagent (IDCR) is used.
3. Protein-to-protein assay uniformity for a set of immunoglobulins, bovine serum albumin and other example proteins. |
|
