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Protein Assay Selection Guide

Choose an assay reagent and appropriate standard to determine protein concentration.

Protein Assay Selection GuideProtein 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.

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Protein Methods Library Home


Overview of Protein Assays

Chemistry of Protein Assays

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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

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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.

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