Rapid, Antibody-Free Detection of Recombinant Proteins on Blots Using Enzyme Fragment Complementation.

Alternative, antibody-free techniques to western analysis of protein blots can offer reduced assay times for routine analysis of expression of recombinant proteins. We have adapted the commercially available enzyme fragment complementation technology to provide a rapid protein detection method for protein blots based on significantly reducing the number of incubation and washing steps used in traditional approaches, and eliminating the requirement for antibodies. In this chapter, we highlight the use of this assay for measuring recombinant protein expressed in mammalian cells for a range of applications, including dot blot screening of large numbers of different cell samples, assessment of protein integrity through detection of degradation bands, and characterization of posttranslational protein modifications such as glycosylation.

Rapid, antibody-free detection of recombinant proteins on blots using enzyme fragment complementation.

Alternative, antibody-free techniques to western analysis of protein blots can offer reduced assay times for routine analysis of expression of recombinant proteins. We have adapted the commercially available enzyme fragment complementation technology to provide a rapid protein detection method for protein blots based on significantly reducing the number of incubation and washing steps used in traditional approaches, and eliminating the requirement for antibodies. In this article, we highlight the use of this assay for measuring recombinant protein expressed in mammalian cells for a range of applications, including dot blot screening of large numbers of different cell samples, assessment of protein integrity through detection of degradation bands, and characterization of post-translational protein modifications such as glycosylation.

A miniaturized glucocorticoid receptor translocation assay using enzymatic fragment complementation evaluated with qHTS.

Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods.

A homogeneous enzyme fragment complementation-based beta-arrestin translocation assay for high-throughput screening of G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between beta-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology.

Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery.

Many cell-based assays interrogating cell pathway activation employ protocols that require microscopic imaging techniques. However, such assays are not in general widely adopted for primary screening. Protein complementation, particularly of enzymes, provides an alternative approach for cell pathway analysis, with a principal advantage that is amenable to high throughput screening using microtiter plate protocols.

In situ reduction of chromium(VI) in heavily contaminated soils through organic carbon amendment.

Chromium has become an important soil contaminant at many sites, and facilitating in situ reduction of toxic Cr(VI) to nontoxic Cr(III) is becoming an attractive remediation strategy. Acceleration of Cr(VI) reduction in soils by addition of organic carbon was tested in columns pretreated with solutions containing 1000 and 10 000 mg L(-1) Cr(VI) to evaluate potential in situ remediation of highly contaminated soils. Solutions containing 0,800, or 4000 mg L(-1) organic carbon in the form of tryptic soy broth or lactate were diffused into the Cr(VI)-contaminated soils.

Distribution of chromium contamination and microbial activity in soil aggregates.

Biogeochemical transformations of redox-sensitive chemicals in soils can be strongly transport-controlled and localized. This was tested through experiments on chromium diffusion and reduction in soil aggregates that were exposed to chromate solutions. Reduction of soluble Cr(VI) to insoluble Cr(II) occurred only within the surface layer of aggregates with higher available organic carbon and higher microbial respiration.