Mobility Shift Analysis of Protein-DNA Complexes by Capillary Electrophoresis

1998 ◽  
pp. 272-291 ◽  
Author(s):  
Jun Xian ◽  
Michael G. Harrington
Keyword(s):  
Capillary Electrophoresis ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Shift Analysis

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

The tumor suppressor p53 regulates its own transcription

1993 ◽  
Vol 13 (6) ◽  
pp. 3415-3423
Author(s):  
A Deffie ◽  
H Wu ◽  
V Reinke ◽  
G Lozano
Keyword(s):  
Consensus Sequence ◽  
Point Mutations ◽  
Direct Interaction ◽  
Mutant P53 ◽  
Tumor Suppressor P53 ◽  
Mobility Shift ◽  
Shift Analysis ◽  
Promoter Deletion Analysis ◽  
Protein Dna Interactions ◽  
Responsive Element

The ability of p53 to suppress transformation correlates with its ability to activate transcription. To identify targets of p53 transactivation, we examined the p53 promoter itself. Northern (RNA) analysis and transient transfection experiments showed that p53 transcriptionally regulated itself. A functionally inactive mutant p53 could not regulate the p53 promoter. Deletion analysis of the p53 promoter delineated sequences between +22 and +67 as being critical for regulation. Electrophoretic mobility shift analysis and methylation interference pinpointed the p53 DNA responsive element. When oligomerized in front of a heterologous minimal promoter, this element was regulated by wild-type p53 and not by mutant p53. Point mutations in the DNA element that eliminated protein-DNA interactions also resulted in a nonresponsive p53 promoter. The DNA element in the p53 promoter responsive to p53 regulation is similar to the p53 consensus sequence. However, we have been unable to detect a direct interaction of p53 with its promoter.

scholarly journals Mobility Shift Affinity Capillary Electrophoresis at High Ligand Concentrations: Application to Aluminum Chlorohydrate–Protein Interactions

ACS Omega ◽  
2018 ◽  
Vol 3 (12) ◽  
pp. 17547-17554
Author(s):  
Nesrine Ouadah ◽  
Claudine Moire ◽  
Fabien Brothier ◽  
Jean-François Kuntz ◽  
Michal Malý ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Protein Interactions ◽  
Affinity Capillary Electrophoresis ◽  
Mobility Shift ◽  
High Ligand

scholarly journals Analysis of multiple forms of nuclear factor I in human and murine cell lines.

1990 ◽  
Vol 10 (3) ◽  
pp. 1041-1048 ◽  
Author(s):  
N Goyal ◽  
J Knox ◽  
R M Gronostajski
Keyword(s):  
Cell Lines ◽  
Binding Activity ◽  
Cross Linking ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Murine Cell ◽  
Growth State ◽  
Factor I ◽  
Nuclear Factor I

Nuclear factor I (NFI) is a group of related site-specific DNA-binding proteins that function in adenovirus DNA replication and cellular RNA metabolism. We have measured both the levels and forms of NFI that interact with a well-characterized 26-base-pair NFI-binding site. Five different NFI-DNA complexes were seen in HeLa nuclear extracts by using a gel mobility shift (GMS) assay. In addition, at least six forms of NFI were shown to cross-link directly to DNA by using a UV cross-linking assay. The distinct GMS complexes detected were composed of different subspecies of NFI polypeptides as assayed by UV cross-linking. Different murine cell lines possessed varying levels and forms of NFI binding activity, as judged by nitrocellulose filter binding and GMS assays. The growth state of NIH 3T3 cells affected both the types of NFI-DNA complexes seen in a GMS assay and the forms of the protein detected by UV cross-linking.

scholarly journals Pyrophosphate in the Binding Reaction Increases the Sensitivity of Mobility Shift Analysis

BioTechniques ◽  
1997 ◽  
Vol 23 (2) ◽  
pp. 248-250 ◽  
Author(s):  
Chris Fenton ◽  
M. Raafat El-Gewely
Keyword(s):  
Mobility Shift ◽  
Binding Reaction ◽  
Shift Analysis

Inhibition of transcription factor assembly and structural stability on mitoxantrone binding with DNA

2010 ◽  
Vol 30 (5) ◽  
pp. 331-340 ◽  
Author(s):  
Shahper N. Khan ◽  
Mohd Danishuddin ◽  
Asad U. Khan
Keyword(s):  
Transcription Factor ◽  
Binding Mode ◽  
Cd Spectroscopy ◽  
Sequence Specificity ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Naked Dna ◽  
Plasmid Nicking Assay ◽  
Modelling Studies ◽  
Mobility Shift Assay

MTX (mitoxantrone) is perhaps the most promising drug used in the treatment of various malignancies. Comprehensive literature on the therapeutics has indicated it to be the least toxic in its class, although its mechanism of action is still not well defined. In the present study, we have evaluated the associated binding interactions of MTX with naked DNA. The mechanism of MTX binding with DNA was elucidated by steady-state fluorescence and a static-type quenching mechanism is suggested for this interaction. Thermodynamic parameters from van 't Hoff plots showed that the interaction of these drugs with DNA is an entropically driven phenomenon. The binding mode was expounded by attenuance measurements and competitive binding of a known intercalator. Sequence specificity of these drug–DNA complexes was analysed by FTIR (Fourier-transform infrared) spectroscopy and molecular modelling studies. CD spectroscopy and the plasmid nicking assay showed that the binding of this drug with DNA results in structural and conformational perturbations. EMSA (electrophoretic mobility-shift assay) results showed that these drug–DNA complexes prevent the binding of octamer TF (transcription factor) to DNA. In summary, the study implicates MTX-induced conformational instability and transcription inhibition on DNA binding.

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