Electrophoretic mobility shift assays

Fluorescein retention measurements are converted to relative amount of fluorescence normalized against protein concentration. Test chemical exposure causing disruption of tight junctions will allow fluorescein molecules to penetrate the comeal tissue and become trapped within its epithelial layer. Thus, fluorescein molecules are used as a permeability tracer and applied in defining comeal epithelial and endothelial permeability (Watsky et al. 1989). FITC-retention provides a quantitative assessment for paracellular leakage 

Comeal organ culture combined with objectively quantifiable assays for comeal epithelial barrier disruption reduces the high variability associated to the subjectively scored Draize Test. The FITC-Dextran retention has been studied as a quantitative evaluation of the comeal epithelial barrier (Lopez et al. 1991) following chemical exposure of bcnzal konium chloride (BAC), Polyquad, and Thimerosal. Sodium dodecyl sulfate (SDS) has also been tested for disruption of the tight junctions via FITC-Dextran retention assay. However, as an objective outcome measure for ocular toxicity, the scoring system is not yet quantitatively comparable for assessment of ocular irritancy to multiple test products. This limitation is similar to surface biotinylation assays. As fluorometry is utilized more extensively in varied laboratories with numerous test chemicals a standardized scoring system can be elicited similar to the familiar Draize Test. 

The concentration of test chemicals applied directly or dissolved in culture media may be modified to test concentration-dependent alterations in epithelial barrier properties. Also different molecular weights of fluorescein may be substituted to alter permeability and retention relationships as a function of the test chemical used. 

Lopez BD, Ubels JL (1991) Quantitative evaluation of the corneal epithelial barrier effect of artificial tears and preservatives. Curr Eye Res 10(7) 645-656 Rein M (2003) The ocular surface barrier function and mechanisms of injury and repair. In Salem H, Katz S (eds) Alternative Toxicological Methods. CRC Press, New York, pp 89-108 

Watsky MA, McDermott ML, Edelhauser HF (1989) In vitro corneal endothelial permeability in rabbit and human the effects of age, cataract surgery and diabetes. Exp Eye Res 49(5) 751—767 

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The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to determine levels of a particular mRNA, nuclear run on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specific elements in the promoter region responsible for the control of expression. Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Reverse transcriptase-polymerase chain reaction and RNase protection assay techniques are used to amplify and quantitate mRNAs, while the electrophoretic mobility shift assay is used to measure binding of a transcription factor to its specific DNA consensus sequence. 

The first relevance on pain was activation of PPARa in the spinal cord of rats with peripheral inflammation (Benani et al., 2004). Electrophoretic mobility-shift assay was employed to compare the DNA-binding activity toward a PPRE. The PPARa isoform was observed to be activated in the rat spinal cord after complete Freund s adjuvant injection, which could elicit hyperalgesia. PPARa was provided as a new player in the molecular modeling of pain pathways, although it was discussed that inhibitors of PPARa activation might be relevant antinociceptive drugs. 

The interactions of the G-quadruplex of human telomere DNA with these newly designed molecules have been examined via CD spectroscopy and electrophoretic mobility shift assay (EMSA). The selectivity between the quindoline derivative and G-quadruplex or duplex DNA has been investigated by... 

To show activation of NF-kB, electrophoretic mobility shift assays are typically performed to look at the specific binding of activated NF-kB to DNA. This technique requires relatively large numbers of cells, is laborious, is not performed in intact cells, and is subject to artifacts. Another typical cellular assay measures translational regulation of gene reporter constructs in transfected cells occurring hours after cellular aetivation. 

In vitro translation is the method of choice for preparing small quantities of protein for analytical purposes and, with the recent availability of commercial reactors and reagents, also for preparative purposes on a modest scale (several mg). Proteins prepared in this way can be analysed by imunoprecipitation or SDS-PAGE they can also be tested for specific biochemical activity, such as enzyme activity or specific DNA binding activity by techniques such as gel electrophoretic mobility shift assays. 

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