The Colorimetric Detection and Quantitation of Total Protein

Protein quantification is an important step for handling protein samples for isolation and characterization, and is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. The methods included in this unit are colorimetric measurements, whose procedures are faster, simpler, and less laborious than those based on estimation of total nitrogen content (vnitbi.2). 

This unit describes four of the most commonly used total protein assay methods. Three of the four are copper-based assays to quantitate total protein the Lowry method (see Basic Protocol 1 and Alternate Protocols 1 and 2), the bicinchoninic acid assay (BCA see Basic Protocol 2 and Alternate Protocols 3 and 4), and the biuret method (see Basic Protocol 3 and Alternate Protocol 5). The fourth is the Coomassie dye binding or Bradford assay (see Basic Protocol 4 and Alternate Protocols 6 and 7), which is included as a simple and sensitive assay, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acidic pH. A protein assay method should be chosen based on the sensitivity and accuracy of method as well as the condition of the sample to be analyzed. 

The four colorimetric methods for the detection and quantitation presented in this unit have withstood the test of time. They are all well-characterized robust assays that consistently work well. The methods were introduced over the past 15 to 50 years. They collectively represent the state of the art for colorimetric detection and quantitation of total proteins in the microgram to milligram range. 

Sometimes the sample contains substances that make it incompatible with any of the protein assay methods. In those cases, some pretreatment of the sample is necessary. 

Each method has its advantages and disadvantages. No one method can be considered to be the ideal or best protein assay method. Because of this, most researchers keep more than one type of protein assay reagent available in their lab. 

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B1.1 The Colorimetric Detection and Quantitation of Total Protein B1.2 Determination of Total Nitrogen... 

In 1976, Marion Bradford introduced the first Coomassie dye-based reagent for the rapid colorimetric detection and quantitation of total protein. The Coomassie dye (Bradford) protein assay reagents have the advantage of being compatible with most salts, solvents, buffers, thiols, reducing substances, and metal chelating agents encountered in protein samples. 

Total protein assays have the advantage of being relatively straightforward compared to molecular-level analyses. Methods with fluorescence-based detection are also highly sensitive, and thus amenable direcdy to DON. Quantitative interpretation for environmental mixtures such as seawater, however, may be problematic for some samples. Most methods react with specific moieties (e.g., coomassie blue binds to lysine and arginine) and thus results obtained can depend on protein composition, size distribution, and even conformation (Sapan et ai, 1999), making the careful choice of calibration standards important. In addition, common components of natural samples, such as humic materials (e.g., Mayer et ai, 1986), carbohydrates (Sapan et ai, 1999), or NH3 may interfere with quantification. Overall, colorimetric methods can be very useful as quick, Hkely semi-quantitative estimates of total protein or peptide. However, potential biases inherent in the mechanism of a specific method should be considered before one is chosen, and appHcation of newer molecular assays (e.g., CBQCA) should be carefully examined in terms of natural sample matrix (Nunn et ai, 2003). 

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