Mobility shift analysis

Fig. 6 Evaluation of phosvitin binding to monoclonal anti-phosphoserine antibody by ACE using mobility-shift analysis and UV detection. Peaks M, internal peptide marker mAb, free monoclonal anti-phosphoserine antibody mAb-hpAg complex, monoclonal anti-phosphoserine antibody complexed with homopolyvalent phosvitin antigen. The buffer contained phosvitin within a concentration from 0 to 60 /xM. (Reprinted with permission from Ref. 27. Copyright 1997 Academic Press.)... 

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.

Fig. 4.1. Mobility shift analysis Mobility shift analysis performed by 4% PAGE in 50 mM Tris-glycine 1 mM EDTA. The mobility shift is induced by the 60 kD polypyrimidine tract binding protein to a 220 nucleotide random labelled pre-mRNA transcript. The experiment shows dimeric binding (2. complex) of the protein at...

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... 

DNA Affinity Purification of BUR-Binding Proteins Measuring BUR-Binding Activity by Southwestern and Gel Mobility Shift Analysis... 

Gel Mobility Shift Analysis with Cloned Inserts... 

Fig. 5 Gel mobility shift analysis of total DNA inserts. Insert DNA was purified and labeled as described. Gei mobiiity shift assay was performed as described with purified bacterially produced SATBl. Lanes 1-4 contain 0, 1,2, and 4 nM of SATBl. Gel mobility shift competition results using a. iO-foid excess of unlabeled wild-type (25), (lane 5) and a. 50-fold excess of unlabeled mutated (24)k (lane 6) are shown.

Fig. 4. DNA-protein interactions in isolated nucleosomal core particles following poly(ADP-ribose)-modification in vitro, Nucleosomal core particles were prepared as described (6, 7) and incubated as indicated. Subsequent gel mobility shift analysis was performed as described (7), using DNA restriction markers of 830,560, and 140 bp.

Fig. 1 Schematic representation of the experimental setups of the mobility-shift method and the Hummel-Dreyer method (A) the vacancy peak method and the vacancy affinity capillary electrophoresis method (B) the equilibrium-mixture method and the frontal analysis method (C) for drug-protein binding analysis. drug protein gg drug-protein complex Q buffer. (Reprinted with permission from Ref. 38. Copyright 1992 Elsevier Science.)...

The decision whether mobility-shift or equilibrium-mixture analysis... 

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. 

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. 

Viadiu H, Aggarwal AK. Structure of BamHI bound to nonspecific DNA a model for DNA sliding. Molec. Cell 2000 5 889-895. Sidorova NY, Muradymov S, Rau DC. Trapping DNA-protein binding reactions with neutral osmolytes for the analysis by gel mobility shift and self-cleavage assays. Nucleic Acids Res. 2005 33 5145-5155. 

Use of a rapid screening method for determination of pKg of candidate drugs by pressure-assisted CE coupled with a photodiode array detector is described. Application of pressure during CE analysis allowed completion of one CE run in less than one minute, and the obtained pH-metric mobility shifts as well as the pH-metric UV spectrum were analyzed by a nonlinear regression fitting software to determine pKa values. The difference between pKa values by this method and by other conventional methods is within 0.25 units for 82 ionic functional groups of 77 drugs. The pK values of 96 compounds in dimethylsulfoxide (DMSO) solution on a 96-well microplate could be measured in one day. Our method provides rapid and accurate determination of pKa values. 

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