Protein concentration assays, general

There are many cases when it may be more convenient, or even necessary to use a colorimetric assay. These methods can be used when a protein s extinction coefficient is not known (or has no Trp/Tyr residues), or is not free of absorbing contaminants. These methods are also useful when trying to nonspecifically determine total protein concentration. In general, they all rely on an exogenous molecule forming a complex with the protein in solution, which leads to an absor-bance/fluorescence signal proportional to the protein concentration. 

For greatest accuracy of the estimates of the total protein concentration in unknown samples, it is essential to include a standard curve in each run. This is particularly true for the protein assay methods that produce nonlinear standard curves (e.g., Lowry method, Coomassie dye-binding method). The decision about the number of standards used to define the standard curve and the number of replicates to be done on each standard depends upon the degree of nonlinearity in the standard curve and the degree of accuracy required of the results. In general, fewer points are needed to construct a standard curve if the color response curve is linear. For assays done in test tubes, duplicates are sufficient however, triplicates are recommended for assays performed in microtiter plates due to the increased error associated with microtiter plates and microtiter plate readers. 

Enzyme assays The amount of enzyme protein present can be determined (assayed) in terms of the catalytic effect it produces, that is the conversion of substrate to product. In order to assay (monitor the activity of) an enzyme, the overall equation of the reaction being catalyzed must be known, and an analytical procedure must be available for determining either the disappearance of substrate or the appearance of product. In addition, one must take into account whether the enzyme requires any cofactors, and the pH and temperature at which the enzyme is optimally active (see Topic C3). For mammalian enzymes, this is usually in the range 25-37°C. Finally, it is essential that the rate of the reaction being assayed is a measure of the enzyme activity present and is not limited by an insufficient supply of substrate. Therefore, very high substrate concentrations are generally required so that the initial reaction rate, which is determined experimentally, is proportional to the enzyme concentration (see Topic C3). 

We generally assess the protein concentration of new chemokine stock solutions by OD280 using the appropriate extinction coefficient for each chemokine or by Bradford assay. Although this is not often practical when very small quantities of chemokine are purchased because 10-20% of the material may be sacrificed for an accurate determination, we have found that the actual protein concentration may vary by as much as 2CM-0% between lots which can significantly effect the reproducibility of chemotaxis assays. 

The most common types of assays employed to quantitate protein concentrations in biological matrices are listed in Table 32.4. Enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunoradiometric assays (IRMAs) require protein-specific antibodies, labeled proteins, or labeled antibodies as reagents, and are generally competitive inhibition assays. Radioimmunoassays measure concentrations by displacing ligands from cell-bound receptors. The most common assay, the... 

Protein concentrations do not necessarily accurately estimate active sites, even when proteins are pure based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Typically, protein concentrations yield upper estimates of active site concentrations. The best method to determine active sites is based on an activity, such as inhibitor binding. Therefore, tight-binding inhibitors are often the best method to determine the active site concentration of an enzyme. As a general rule, the best estimates of active site concentration come when the maximal enzyme concentration used in the assay is at least five- to 10-fold greater than the inhibitor IC50 value. 

Nephelometric methods in general are more sensitive than turbidimetric assays and have an average lower limit of detection of 0.1 to 10 mg/L for a serum protein. Lower detection limits are obtained in fluids, such as cerebrospinal fluid and urine, because of their lower lipid and protein concentrations, which results in a better signal-to-noise ratio. In addition, for low molecular weight proteins (e.g., myoglobin, MW 17,800), assay detection Emits can be lowered using a latex-enhanced procedure based on antibody-coated latex beads. ... 

Assay Generally HPLC is sufficient for assay, identification, and impurities Generally overall protein concentration, plus at least one specific bioassay and one or more assays showing binding correlated with clinical experience during development... 

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