Protein Purity and Quantity Determination

It is a crucial task to accurately determine the purity and quantity of the target protein because that the results may subsequently used in other calculations include enzyme activity analysis. There are a lot of analysis methods to select, and you need to aware that the absolutely accurate results can not be achieved by a single protein assay method and each assay has its own advantages and disadvantages.

The Lowry Assay
The Lowry Assay is based on two reactions. The first one is to form a copper ion complex with amide bonds, leading to reduced copper in alkaline solutions. The second one is to reduce the Folin-Ciocalteu reagent bytryptophan and tyrosine residues and by the reduced copper ion complex with amide bonds. The detectable range for Folin-Lowry is from 500 to 750 nm. This assay is an endpoint method since it provides a stable result by fitting to a standard curve. However, it is relatively sensitive and incompatible with a wide variety of common chemicals such as EDTA, DTT, Tris, etc.

The Bicinchoninic Acid (BCA) Assay
The BCA assay is based on the measurement of the purple colored protein-bound copper-BCA chelates. This assay is similar to the Lowry assay but with the replacement of Folin-Ciocalteu reagent to BCA reagent. A strong absorbance at 562 nm will be detected when BCA and copper ion form a complex. The advantage of this assay is that it does not interact with detergents, making it a better choice compared with Bradford assay in case that the sample contains detergent contaminants.

The Bradford Assay
The Bradford assay is one of the most used protein assay since it is elegantly simple, inexpensive, fast, stable, and sensitive. The principle of Bradford assay is that residues such as arginine, tyrosine, and histidine can direct bind to Coomassie brilliant blue dye. The solution color changes from red to blue after the binding, and the absorbance changes from 465 nm to 595 nm. You can compare the sample to a standard curve. However, the Bradford assay only works for proteins larger than 3 kDa, and it is not suitable for protein samples contain detergents.

The Ultraviolet Absorbance Measurement
Protein concentration measurement based on the ultraviolet absorbance is simple and quick, however, it may yield inaccurate results. It usually estimates the amount of protein by ultraviolet absorbance measurement of tyrosine and tryptophan in protein sample at 280 nm. UV measurement is very sensitive to ionic strength and pH, and it highly depends on the varying amount of tyrosine and tryptophan amino acids, making this methods sometimes unreliable.

The Kjeldahl Assay
The Kjeldahl assay is one of the oldest protein assay. It based on the nitrogen measurement in a protein sample. Advantages of the Kjeldahl assay are its good reproducibility, high precision, and universality, making it still the major assay to estimate the protein amount in foods. Disadvantages of the Kjeldahl assay are time-consuming, considerably hazardous, and sample-consuming, making it very impractical.

Creative Biostructure is the expert in protein quality control as well as in protein expression and purification. We have established this resourceful technical support platform to solve your problems. Please see the related services for more information and feel free to Contact Us.

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