Biomedical Engineering Reference
In-Depth Information
Similar to cell-based assays, cell-free assays offer their own significant advan-
tages, which have made them very popular. Although some cell-free assays employ
cell lysates, a great majority of them are biochemical assays that are based on purified
macromolecules present in a buffer of optimal composition, greatly reducing non-
specific binding of compounds. Biochemical assays are extremely attractive, as they
provide easily interpretable quantitative results, unhindered by compound membrane
permeability or nonspecific protein binding. On the other hand, since biochemical
assays employ highly diluted solutions of pure protein preparations, they frequently
face a challenge of keeping the intracellular proteins in their native state and combat-
ing undesired protein unfolding, oxidation, or nonspecific binding to organic polymer
surfaces.
12.3.3 Common Biochemical Screening Methods
In addition to being classified into cell-based and cell-free assay groups, most assays
could be further affiliated with either binding or functional assays. Both of these
approaches are generally applicable to either cell-based or biochemical assays. Quite
frequently, the name binding refers to assays that employ displacement of a known
ligand or a protein partner from a binding complex with the target of interest. To
distinguish them from assays that actually detect compound binding, the term dis-
placement assays is more appropriate for describing this type of assays.
Functional biochemical assays most frequently involve a single catalytic principal
enzyme in reaction mixtures. However, some assays are built to reconstitute a mul-
tistep pathway. In some cases, selection of this approach is dictated by an inability
to generate a substrate for a protein of interest; in this case, an enzyme preceding
the target of interest in the native pathway is utilized to generate the substrate. An
additional benefit of employing a pathway assay is that it provides an opportunity of
targeting multiple potential targets in a single assay. Examples of successful path-
way assays are provided by ubiquitination, SUMOylation, and neddylation assays
performed at the CPCCG [11].
In a functional biochemical assay, a compound's binding is gauged though changes
in substrate binding or catalytic efficiency of the active site. The enzyme catalytic
activity is usually monitored directly through generation of a product or consumption
of a substrate. In some cases, a product of the reaction is converted further in a
coupled assay, leading to the generation of a detectable moiety. For example, many
metabolic reactions could be coupled with those consuming or generating NAD(P)H
or ATP. NAD(P)H is detectable directly through absorbance at 340 nm or fluores-
cence at 460 nm; alternatively, its concentration could be assessed by using bacterial
luciferase to provide luminescent signal or a resazurin/diaphorase reaction, leading
to the generation of the fluorescent product resorufin. The concentration of ATP can
be assessed using firefly luciferase to generate luminescence. Other reaction types
dealing with protein modification and not easily coupled with a detectable chem-
istry (e.g., dephosphorylation or proteolysis) are commonly monitored with the help
of antibodies that recognize the product or via a mock reaction with an artificial
fluorogenic or luminogenic substrate.
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