Electrophoretic Mobility Shift Assay EMSA

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... 

A wealth of data demonstrates that NF-kB is activated und conditions of inflammation and oxidative stress (Czaja 2007 Gloire et al. 2006). Consistent wilh this, studies with PPARa activators demonstrate linkages between oxidative stress and NF-kB activation. Activation is usually assessed by the ability of nuclear NF-kB (usually a heterodimer composed of p50 and p65 subunits) to bind to a NF-kB response element in an electrophoretic mobility shift assay (EMSA). In whole liver of both rats and mice, activity of NF-kB was increased by PPARa activators including WY-14,643, ciprofibrate, and gemfibrozil bnt not nafenopin (Table 17.1). The fact that nafenopin did not induce NF-kB may be due to differences in the EMSA procedures carried out by that lab (Menegazzi et al. 1997 Ohmura et al. 1996). 

The electrophoretic mobility shift assay (EMSA), also called the gel-shift or band-shift assay, is more useful than the footprinting assay for quantitative analysis of DNA-binding proteins. In general, the electrophoretic mobility of a DNA fragment is reduced when it is complexed to protein, causing a shift in the location of the fragment band. This assay can be used to detect a transcription factor in protein... 

Sequence-specific DNA binding activity can be assessed by several methods two of the most sensitive and useful are DNA footprinting and electrophoretic mobility shift assays (EMSA). These procedures have been utilized to identify and purify various DNA binding proteins required for transcription, such as the C/EBP protein, which binds the 5e-CCAAT-3c sequence (Fig. 24.5). 

Protein-DNA interactions can be characterized experimentally by the DNA footprinting technique and the electrophoretic mobility shift assay (EMSA). (a) In vitro DNAse I footprinting (b) EMSA. Reactions contained an end-labeled radioactive fragment (a) or oligonucleotide (b) incubated with the zinc-finger DNA binding domain of the... 

Many inflammatory cytokines including IL-8 are regulated at transcriptional levels, and a variety of transcription factors such as nuclear factor-K B (NFkB) and activator protein-1 (AP-1) play important roles in such processes. Abe and coworkers [71] demonstrated that CAM repressed TNF-a-induced AP-1 activation in human bronchial epithelial cells. We studied the effect of EM and CAM on the phorbol myristate acetate (PMA)-induced activation of NFkB and AP-1. Pretreatment of EM and CAM before the PMA treatment showed an inhibitory effect on both of the transcription factors as assessed by electrophoretic mobility shift assay (EMSA) (Fig. 14) [20]. In contrast, the macrolides showed no effect on the activation of cyclic AMP-responsive element binding protein (CREB), suggesting that the suppressive effect on some transcription factors is specific. We further evaluated the effect of EM on the phosphorylation of inhibitor of NFkB (IkB), which is a crucial step for transactivation of NFkB. EM did not influence the phosphorylation processes in vitro (Okazaki et at, unpublished data, January 2001). These data suggest that EM acts at the process of nuclear translocation of... 

The first studies to address whether STAT proteins can be activated in neutrophils were published 5 months apart in 1995-1996. In one of them, Tweardy et al. [32] investigated the effect of several neutrophil agonists on this response. Whole-cell extracts from neutrophils disrupted by freeze-thaw cycles were analyzed in an electrophoretic mobility shift assay (EMSA) using a human serum-inducible element (hSIE/m67) oligonucleotide probe. A specific complex was induced in cells treated with G-CSF, but could not be supershifted using antibodies raised against individual STAT proteins. The authors concluded that granulocytes express a novel STAT-like protein, which they called STAT-G... 

Further investigations focused on evaluating the three steps of IFN-y-stimulated class II expression signal transduction and transcription factor activation, CIITA expression, and class II transcription in CMV-infected cells. Ribonuclease protection assays (RPA) and RT-PCR experiments reveal that neither MHC class II nor CIITA RNA is upregulated in response to IFN-y treatment in HCMV-infected ECs at 72h after infection (Miller et al. 1998). Moreover, electrophoretic mobility shift assays (EMSA) demonstrate that STAT-1 homodimers are not... 

Heilman LM, Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc 2(8) 1849-1861.doi 10.1038/nprot.2007.249... 

The rat hepatic cytosol fraction was prepared as described previously (7). The cytosol fraction (4.0 mg/ml protein) was incubated with various concentrations of green tea extracts 10 min prior to addition of 1 nM TCDD (AccuStandard, New Heaven, CT, USA) at 20°C for 2 h to lead transformation of AhR. Transformed AhR was detected by electrophoretic mobility shift assay (EMSA) using a P-labeled dioxin responsive element (DRE) oligonucleotide probe as described previously (7). 

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