Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay

1989 ◽  
Vol 10 (5-6) ◽  
pp. 366-376 ◽  
Author(s):  
Michael G. Fried
Keyword(s):  
Dna Interaction ◽  
Interaction Parameters ◽  
Mobility Shift ◽  
Electrophoresis Mobility Shift Assay ◽  
Mobility Shift Assay

scholarly journals Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

2005 ◽  
Vol 70 (5) ◽  
pp. 705-712 ◽  
Author(s):  
Miroslava Vujcic ◽  
Natasa Terzic ◽  
Aleksandra Ristic-Fira ◽  
Dusan Kanazir ◽  
Sabera Ruzdijic
Keyword(s):  
Dna Interaction ◽  
Binding Activity ◽  
Regulatory Proteins ◽  
Nuclear Proteins ◽  
Middle Aged ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Supershift Assay ◽  
Mobility Shift Assay ◽  
Oligonucleotide Analogues

Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing. .

Electrophoresis Mobility Shift Assay

BIO-PROTOCOL ◽  
2014 ◽  
Vol 4 (7) ◽  
Author(s):  
Masaru Nakata ◽  
Masaru Ohme-Takagi
Keyword(s):  
Mobility Shift ◽  
Electrophoresis Mobility Shift Assay ◽  
Mobility Shift Assay

scholarly journals Integration Host Factor Is Required for RpoN-Dependent hrpL Gene Expression and Controls Motility by Positively Regulating rsmB sRNA in Erwinia amylovora

2016 ◽  
Vol 106 (1) ◽  
pp. 29-36 ◽  
Author(s):  
Jae Hoon Lee ◽  
Youfu Zhao
Keyword(s):  
Gene Expression ◽  
Type Iii Secretion ◽  
Erwinia Amylovora ◽  
Integration Host Factor ◽  
Host Factor ◽  
Mobility Shift ◽  
Electrophoresis Mobility Shift Assay ◽  
Mobility Shift Assay ◽  
Nucleoid Structure

Erwinia amylovora requires an hrp-type III secretion system (T3SS) to cause disease. It has been reported that HrpL, the master regulator of T3SS, is transcriptionally regulated by sigma factor 54 (RpoN), YhbH, and HrpS. In this study, the role of integration host factor (IHF) in regulating hrpL and T3SS gene expression was investigated. IHF is a nucleoid-associated protein that regulates gene expression by influencing nucleoid structure and DNA bending. Our results showed that both ihfA and ihfB mutants of E. amylovora did not induce necrotic lesions on pear fruits. Growth of both mutants was greatly reduced, and expression of the hrpL and T3SS genes was significantly down-regulated as compared with those of the wild type. In addition, expression of the ihfA, but not the ihfB gene, was under auto-suppression by IHF. Furthermore, both ihfA and ihfB mutants were hypermotile, due to significantly reduced expression of small RNA (sRNA) rsmB. Electrophoresis mobility shift assay further confirmed that IHF binds to the promoters of the hrpL and ihfA genes, as well as the rsmB sRNA gene. These results indicate that IHF is required for RpoN-dependent hrpL gene expression and virulence, and controls motility by positively regulating the rsmB sRNA in E. amylovora.

Interaction of long telomeric DNAs with macrocyclic hexaoxazole as a G-quadruplex ligand

MedChemComm ◽  
2013 ◽  
Vol 4 (1) ◽  
pp. 260-264 ◽  
Author(s):  
Keisuke Iida ◽  
Gen Tsubouchi ◽  
Takahiro Nakamura ◽  
Satoki Majima ◽  
Hiroyuki Seimiya ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Living Cells ◽  
Dna Melting ◽  
Mobility Shift ◽  
Electrophoresis Mobility Shift Assay ◽  
G Quadruplex ◽  
Mobility Shift Assay ◽  
Circular Dichroïsm

The interactions of long telomeric DNAs, which mimic telomeres in living cells, with a macrocyclic hexaoxazole ligand L2H2-6OTD (2) were investigated by means of electrophoresis mobility shift assay, circular dichroism (CD) titration analysis, and DNA melting measurements.

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