Gel retardation assay

The 5 LTR has been extensively characterized in vitro, and binding sites for several cellular transcription factors have been identified using DNase I in vitro footprinting and gel retardation assays (Fig. Ic) (see for reviews Roebuck and Saifuddin, 1999 Pereira et ai, 2000 Rohr et al, 2003). 

We have used gel retardation assays to study proteins binding to the ARE. We used a probe consisting of the 42 base ARE. When the probe was multimerised in four or six tandem copies, protein, AREF (ARE Factor) from nuclear extracts of anaerobically induced suspension cultures was bound to the probe and showed a ladder of retarded bands (Fig. 2). This ladder of bands was completed specifically by the 4x ARE probe but not by a probe containing four copies of the octopine synthase enhancer element or pUC DNA even in 100-fold excess. Probes which contained one or two copies of the ARE did not show any binding. The need to multimerise the probe presumably results either from low affinity of the protein for the DNA or low abundance of AREF in nuclear extracts. 

For preparation of lipoplexes, first of all, concentrated stocks of nncleic acid and cationic liposome are prepared and stored at optimal condition (-80°C to 4°C). Then, the concentrated stocks are diluted with required volume of diluent according to desired N/P ratio. An example calculation of N/P ratio is presented. Then, the diluted nucleic acids and liposomes are mixed and stand at room temperature for desired time to allow the lipoplex formation. The formation and stability of complexes can be assessed with the gel retardation assay. 

Visualize and photograph the agarose gel stained with ethidium bromide under transillumination at 300 nm (UV light). The nucleic acid-lipoplex should remain inside the well, while the free or weakly bound nucleic acid should run in the gel. An example of gel retardation assay is shown in Fig. 2. 

Fig. 2. Gel retardation assay of naked siRNA and siRNA-LipoTrusF lipoplexes formed spontaneously in a 2% agarose gel. The amount of cationic lipid was 9.6 pM. The siRNA doses correspond to the cationic lipidVsiRNA charge ratio of 0.95,1.90,3.81 and 7.62, respectively. LipoTmsF is constituted of dioleoylphosphatidylethanolamine, cholesterol and the cationic lipid 0,0 -ditetradecanoyl-/V-(a-trimethyl ammonioacetyl) diethanolamine chloride (DC-6-14) in the molar ratio of 0.75/0.75/1.00...

T7 RNA polymerase incorporates biotinylated nucleotide analogues efficiently (Fenn and Herman, 1990). They noted that this enzyme did not discriminate significantly between UTP and BIO-4-UTP, but its turnover number was reduced from 130 to 28 pmol/min and the from 32 to 77 p.M for UTP and BIO-4-UTP, respectively). T3 RNA polymerase incorporates BIO-ll-UTP most efficiently (3 X SP6 and 2 X T7 RNA polymerase) (D Alessio, 1985). Fenn and Herman (1990) separated the nucleotides obtained after alkaline hydrolysis of the transcripts by reverse-phase HPLC. Gel retardation assays (Theissen et al., 1989) are simpler alternatives (change in mobility of transcripts on polyacrylamide if streptavidin is bound to biotin moieties) although RNA with secondary structures may not allow the streptavidin to bind under native conditions. 

The mobility shift assay (or gel retardation assay) is based on the difference in mobility between naked RNA and proteins-RNA complexes when migrating in a native polyacrylamide gel. The increased size of the complex and the usually low or positive charge of nucleic acid binding proteins give rise to the difference in mobility. 

At first, the pH-responsive complexation of LMWSC to ODN was investigated by a gel retardation assay (as shown in Figure 10.3a,b). The negatively charged DNA moves to anode under the... 

Fig. 3 Gel retardation assay for Xenopus TFIIIA binding to 5S, P-labeled RNA using 6% native polyacrylamide gel. A, Thionein inhibition of TFIIIA binding to 5S RNA. Lane I, no TFIIIA lane 2, with TFIIIA (final concentration, 300nAf) lanes 3-9, addition of 32, 16, 8, 4, 2, 1, and 0.5 M thionein, respectively lane 10, 32pM Zny-thionein. B, Restoration of TFIIIA binding to 5S RNA in the presence of thionein by addition of zinc. Lane I, no TFIIIA lane 2, with TFIIIA lane 3, addition of micromolar thionein lane 4, addition of 32pM thionein and then 130pM zinc. 5S, 5S RNA 7S, TFIIIA-5S RNA complex (7S RNP). (From Zeng et al. 1991b)...

Fig. 5 Gel retardation assay with HeLa cell nuclear extract. A, Thionein (apoMT) inhibition of binding of Spl to P-labeled cognate DNA probe. Lane 7, no extract lane 2, with extract lane 5, competition with 1000-fold excess unlabeled Spl binding sequence lanes 4-10, addition of 13.6, 6.8, 3.4, 1.7, 0.85,...

Fluorescence resonance energy transfer (FRET) was used by Lee et al. (2008) to monitor the dissociation of the chitosan-DNA complex when samples of chitosan with different molecular weights were tested. The chitosan-DNA complex formation was monitored using dynamic light scattering and a gel retardation assay. With HMW chitosans, more condensed complexes were obtained at various ratios of chitosan to DNA. Plasmid DNA and chitosan were separately labeled with quantum dots and Texas Red, respectively. Because quantum dots have high extinction coefficients, they served as excellent FRET donors to excite proximal FRET acceptors. Thanks to this property, the chitosan-DNA complexes were visualized inside the cells. 

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