Denaturing gel electrophoresis

FIGURE 9.6 The loss of cross-linking activity of an aqueous solution of QMP11 (100 pM) in the presence (black) and absence (gray) of dA (20 mM). Cross-linking activity was measured at the indicated times by addition of duplex DNA (3 pM) and subsequent analysis by denaturing gel electrophoresis. Source Adapted from Angew. Chem. Int. Ed. 2008, 47, 1291-1293.69... 

Bprresen, A.-L. (1996) Constant denaturant gel electrophoresis (CDGE) in mutation screening. In Pfeifer, G.P. (ed.) Technologies for Detection of DNA Damage and Mutations. Plenum Press, New York. 

Myers, R.M., Maniatis, T. and Lerman, L.S. (1987) Detection and localization of single base changes by denaturing gel electrophoresis. Methods in Enzymology 155, 501-527. 

The RPA is a sensitive method for quantifying specific RNAs from a mixture of RNAs. This is achieved using a small-volume hybridization of an RNA probe to the RNA under study. Unhybridized probe and sample is then digested with RNAses and the protected probe fragment is visualized after denaturing gel electrophoresis. Commonly, the probe is radiolabeled for maximum sensitivity. Following is a method for RPA detection of R-luc-4 sites and F-luc mRNA. 

Non-denaturing gel electrophoresis Denaturing (SDS) gel electrophoresis Two-dimensional electrophoresis Capillary electrophoresis Peptide mapping HPLC (mainly RP-HPLC)... 

SDS-PAGE analysis of purified FDH revealed two subunits and, in combination with native PAGE, suggested a a2 i structure. Selenium was not released from the protein during denaturing gel electrophoresis and was... 

Another selenium-containing molybdenum hydroxylase that has been isolated from Clostridium barkeri (identical to Eubacterium barkeri) is nicotinic acid hydroxylase (NAH). Clostridium barkeri was isolated initially as a fermentor of nicotinic acid and thus NAH is a key enzyme in the efficient fermentation of nicotinic acid as a source of carbon and energy. NAH contained selenium when purified from cells labeled with Se-selenite. However, this label was lost during denaturing gel electrophoresis and also on heating of the enzyme (Dilworth 1982). Exhaustive analysis of selenium-labeled alkylation products of NAH under various conditions revealed selenium was bound as a labile cofactor (Dilworth 1982), and not as seleno-cysteine. This report was the first to describe a selenium-dependent enzyme that did not contain selenium in the form of selenocysteine. 

Constant denaturing gel electrophoresis is similar to DGGE, but uses a constantly denaturing gradient to achieve the goal of separating characteristically differently charged molecules on a gel. 

By combining denaturing gel electrophoresis (SDS-PAGE) and LA-ICP-MS an analytical technique was developed for the speciation of selenoproteins in Se contaminated wildlife (waterfowl embryo and fish ovary collected from Se contaminated sites in Wyoming and the San Joaquin Valleys, CA, respectively).72 Polatajko et al. reported on the state of the art of selenium speciation in biological samples.73... 

All RNAs that contain 2AP must be chemically synthesized. The phosphoramidite form of 2AP is commercially available from Glen Research (Sterling, VA, USA) for in-house synthesis. Dharmacon (Boulder, CO, USA) and IBA (Gottingen, Germany) do chemical synthesis of RNAs with 2AP. Both suppliers offer protected and deprotected forms of the oligos if money is no object, these suppliers will purify the RNA by denaturing gel electrophoresis or HPLC. RNAs with 2AP require no special handling beyond normal RNA care. 

We probed the reactivity of the carboxylates using a fluorescent carboxylate-selective chemical dye, N-cyclohexyl-N -(4-(dimethylamino)naphthyl)carbodiimide (NCD4). Using UV-visible spectroscopy, native gel electrophoresis, and denaturing gel electrophoresis, covalent modification with the dye was confirmed (Figure 9.5). The latter showed that the dye was attached to both the S and the L subunit, an expected observation, as the structural data suggested carboxylates on both subunits. A quantification of the number of bound dye molecules could not be achieved owing to the instability of the dye in aqueous solvent a reliable extinction coefficient could not be determined. 

The extent of ADP-ribosylation is checked by separating modified and unmodified actin using non-denaturing gel electrophoresis, whereas SDS gel electrophoresis is not efficient. ADP-ribosylation changes the isoelectric point of actin to more acid values, resulting in increased migration. This method is also appropriate to study double or triple ADP-ribosylation of actin, e.g., in the presence of iota toxin and/or turkey erythrocyte transferase (Just ef al., 1995). The latter transferase modifies actin even at two arginine residues. 

Hovig, E., Smith-Sorensen, B Brogger, A., and Bprresen, A.-L. (1991) Constant denaturant gel electrophoresis, a modification of denaturing gradient gel electrophoresis, in mutation detection. Mutation Res. 262, 63-71. 

Condie, A., Ecles, R., Bprresen, A.-L., Coles, C., Cooper, C., and Prosser, J. (1993) Detection of point mutations in the p53 gene comparison of single-strand conformation polymorphism, constant denaturant gel electrophoresis, and hydroxylamine and osmium tetroxide techniques. Human Mutat. 2, 58-66. 

Borresen AL, Hovig E, Smith-Sorensen B, Malkin D, Lystad S, Andersen TI, et al. Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations. Proc Nad Acad Sci U S A 1991 88 8405-9. 

In vitro synthesised RNA is labelled by the inclusion of [a32P]UTP (3000 Ci/mmol 10 mCi/ml) in a standard 10 pi reaction as described in Section 2.3.1 and purified by denaturing gel electrophoresis or through a spin-column (see Section 2.1.2). 

The analysis and quantification of splicing products are conveniently done by denaturing gel electrophoresis. An example of... 

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