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Protein assay: principle and application

Protein assay represents one of the most fundamental and yet important techniques of life science research. Since several protein assay protocols have been
developed over the past several decades that differ widely in their assay principle, sensitivity, reproducibility, and methodology, the criteria for choice of a protein
assay can be based on many factors such as sample amount, sample size, convenience, presence of interfering agents, robustness, and reproducibility. However,
unlike nucleic acid assay, which can be easily and accurately performed based on UV light absorption at 260 nm, protein assay almost invariably relies on a
color-changing chemical reaction and the use of a protein standard, usually purified bovine serum albumin (BSA) or immunoglobulin G (IgG). Understandably, this
means different protein assay protocols can result in various results because different proteins have different amino acid compositions that can differentially affect
the chemical reaction and/or light absorption.

Four major protein assay protocols have been extensively used in the literature: Lowry assay, Biuret assay, Bradford assay, and BCA assay. First documented in
1951, Lowry assay was an often-cited general use protein assay for some time, but has been largely replaced by more robust assay protocols. Lowry assay takes
advantage of the observation that under alkaline conditions the divalent copper ion forms a complex with peptide bonds and becomes reduced. Monovalent copper
ion and the radical groups of tyrosine, tryptophan, and cysteine react with Folin-Ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid)
to produce an unstable product that becomes reduced to molybdenum/tungsten blue. The concentration of the reduced Folin reagent is measured by absorbance
at 750 nm. This method is relatively tedious and not quite sensitive compared to other protein assay methods. Biuret assay is similar to Lowry assay in that it is
also based on formation of colored coordination complexes in an alkaline solution between divalent copper ion and peptide bonds. However it involves a single
incubation of 20 min, reads at 550 nm, and consumes much more material. The assay is thus a general protein assay for batches of protein samples for which
yield is not a problem.

Bradford assay, first documented by Marion Bradford in 1976, is fast and fairly accurate. The assay is based on the observation that the absorbance maximum for
an acidic solution of Coomassie Brilliant Blue G-250 exhibits a dramatic shift from 465 nm to 595 nm upon dye binding to protein. It is thought that both
hydrophobic and ionic forces stabilize the anionic form of the dye, causing a visible color change. One of the two protein assay systems offered by us is formulated
from Bradford assay, and is designed to streamline the procedure for determining concentration of soluble proteins. Unlike those used in other commercial protein
assay kits, this
Protein Assay kit does not require dilution or filtration of the assay reagent prior to the assay, thus saving time and enhancing assay consistency.
This protein assay system has a linear working range of 20 - 200 ug/ml soluble proteins. It should be noted that the presence of detergents in protein samples can
cause interference and reduce assay sensitivity in the Bradford-based protein assay.

Detergent-containing protein samples can perhaps be best analyzed by BCA assay, which combines two famous reactions- the Biuret reaction and the Smith
reaction. The Biuret reaction depicts the reduction of cupric ion (Cu2+) to cuprous ion (Cu1+) by protein in an alkaline solution as described above. The Smith
reaction depicts the chelation of each Cu1+ ion by two molecules of bicinchoninic acid (BCA), resulting in an intense purple-colored product that absorbs light at
562 nm. This detergent-compatible
Protein Assay Kit/DC is however incompatible with most divalent ions. One advantage of this assay system is that it has a
broad linear working range of 20 - 2000 ug/ml proteins, making it an ideal assay for many general biochemical applications.
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