Electrophoretic mobility shift analysis

In a recent study, Mandola et al. showed that the 28 bp TSER tandem repeats contain elements that bind upstream stimulating factor (USF), and also that ligand binding by USF-1 and USF-2 enhances the transcriptional activity of the TS gene (Fig. 2) (42). Electrophoretic mobility shift analysis has shown that the presence of a G-to-C single nucleotide polymorphism (SNP) within the second repeat of the 3R allele leads to decreased ability of upstream stimulatory factor (USF) to bind within the repeat and therefore sequentially result in decreased transcriptional activity of the 3R TS gene variant (42). 

Abbreviations used EMSA, electrophoretic mobility shift analysis IMPase 1, inositol monophosphatase 1 inositol synthase, A/vo-inositol 3-phosphate synthase NCBI, National Center for Biotechnology Information PCR, polymerase chain reaction Rb, retinoblastoma protein tss, transcriptional start site IP3, Inositol trisphosphate DAG, diacyl glycerol FISH, fluorescent in situ hybridization. 

Figure 2. Electrophoretic mobility shift analysis of TFl binding to (A) perfect duplex or (B) duplex with two 4-nt loops separated by 9 bp. Protein concentrations are indicated below.

The abbreviations used are HMG-CoA, 3-hydroxy-3-melhylglutaryl-CoA CAT, chloramphenicol acetyltransferase PPAR, peroxisome proliferator-activated receptor PPRE, peroxisome proliferator-responsive element NRRE, nuclear receptor responsive element RXR, retinoid X receptor hRXRa, human 9-cis-retinoic acid receptor a mPPARa, mouse peroxisome proliferator-activated receptor a COUP-TP, chicken ovalbumin upstream-promoter transcription factor, HNF-4, hepatocyte nuclear factor 4 EMSA, electrophoretic mobility shift analysis tk, thymidine kinase NEFA, nonesterified fatty acids... 

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 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... 

Comparisons of the fimctional SPRR promoter regions has allowed the rapid identification of both conserved and of novel regulatory elements that appeared after gene dupHcation. In vivo analysis has been used to indicate their functionality. Competitive electrophoretic mobility shift assays has confirmed their presence and aided in reveahng their identity. 

In the Drosophila circadian clock, three proteins are rhythmically phosphorylated throughout the circadian cycle PER, TIM and dCLK. The electrophoretic mobility of these three proteins all undergo changes during the circadian day by Western analysis. In all cases, phosphatase treatment reduced or eliminated the slower migrating bands, suggesting that the mobility shifts are due to phosphorylation (Edery et al 1994, Zeng et al 1996, Lee et al 1998). While there is evidence that DBT phosphorylates PER and SGG phosphorylates TIM, it is possible that other kinases phosphorylate these proteins as well. TIM, for instance, is phosphorylated by a tyrosine kinase before it is ubiquitinated and... 

Cycling protein phosphorylation plays a role in the mammalian circadian clock as well. PERI, PER2 and BMAL all show temporal changes in electrophoretic mobility that are eliminated by phosphatase treatment (Lee et al 2001). Although these PER phosphorylations are likely to reflect CKl activity they may not be the only clock-related substrates of this enzyme family. CRYl and CRY2, for instance, can be phosphorylated by CKls in vitro when present in a CRY/PER/CKle complex (Eide et al 2002). Two isoforms of mammalian CLOCK (orthologue of Drosophila CLK) also appear to be phosphorylated, resulting in mobility shifts by Western analysis (Lee et al 2001). The kinase(s) responsible for CLOCK phosphorylation is (are) unknown. 

Because conformational changes in RNA or short DNAs typically cause small changes in electrophoretic mobility, analysis of nucleic acid folding requires careful optimization of electrophoresis conditions. By contrast, protein—nucleic acid interactions are typically easier to analyze by native PAGE because the molecular weight and positive charge of the protein produces a relatively large shift in gel mobility. 

Treatment of brain CBP-II with alkaline phosphataes resulted in the loss of calcium binding activity and a marked shift to electrophoretic mobility. This indication of protein-bound phosphate in brain CBP-II was confirmed by chemical analysis which indicated the presence of ten moles of phosphate per mole of protein. No carbohydrate or lipid moieties were detected. 

The analysis of microbial proteins to locate suitable molecular markers has also been used to discriminate different virus and bacterial isolates. Qualitative changes in electrophoretic mobility can reflect fundamental changes in gene sequence. For example, a characteristic shift in the mobility of the poliovirus structural protein, VPS, correlated not only with an amino acid substitution but also a mutated antigenic epitope (Cash, 1988 Cash,... 

Modification by peptidyl moieties does not alter drastically the electrophoretic mobility of the ribosomal proteins, and despite small shifts in position modified proteins can be identified by two-dimensional gel electrophoresis. Immunoprecipitation has also been applied in the analysis of the modified proteins. - ... 

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