Question:
What are the most commonly used protein assays?
The Protein Man Says:
Most laboratory researchers often face the challenge of selecting the most suitable assay to get an accurate estimation of the protein concentration in their sample. They definitely have to get it right since protein quantitation is generally considered as one of the most important steps prior to processing protein samples for isolation, separation and analysis. To do this, they need to understand the advantages and limitations of each protein assay.
Protein Estimation Assays
There are a number of protein assays that can be used for the determination of protein concentration in a solution. Basically, there are two general types of protein assays – absorbance assays and colorimetric assays.
Absorbance assays give fast results and are more convenient to use since they do not require any protein standards, additional reagents and/or incubation. The amino acids with aromatic side rings (tyrosine, tryptophan and phenylalanine) absorb ultraviolet light at 280 nm while peptide bonds absorb ultraviolet light at 200 nm. However, direct UV-light absorbance assays have several disadvantages that make them impractical for use in most laboratory settings, one of which is the requirement for quartz cuvettes.
The concentration of the protein is calculated according to the following formula which is an approximation to the data of Warburg and Christian for a 1cm pathlength. The absorbance at 260nm accounts for nucleic acid interference. (Warburg, O. and Christian, W. Biochem. Z. 310: 384 (1941)):
Concentration (mg/ml) = (1.55 x A280) - (0.76 x A260)
Colometric protein assays, on the other hand, are more popularly used in most proteomic research applications. In using these assays, protein is added to the reagent, producing a color change proportional to the amount added. The result is then compared to a standard curve to determine the concentration of protein in the sample.
There are two types of colorimetric assays - (1) those that are based on protein-copper chelation and the detection of the reduced copper ions contained in the sample and (2) those that bind with a protein dye, such as Coomassie dyes, and produce a color change which is proportional to the amount of protein added to the solution.
We will take a more in-depth look at some of the most commonly used colorimetric protein assays in our next post so please watch out for it.
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