A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system

Garner, Mark M.; Revzin, Arnold

doi:10.1093/nar/9.13.3047

Cited by 1,966 publications

(1,006 citation statements)

References 34 publications

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“…To examine whether the quantitative and qualitative variations of Jun and Fos protein levels induced by the mutant Ras proteins modi®ed AP1 activity, we measured the AP1 DNA binding activity in nuclear extracts by gel retardation assays (Garner and Revzin, 1981;Piette et al, 1988). The level of AP1 DNA Figure 4 Level and composition of AP-1 binding complexes.…”

Section: Ras Enhances Ap1 Activitymentioning

confidence: 99%

Transformation by ras modifies AP1 composition and activity

et al. 1997

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The Ras proteins play a central role in regulating cell growth and their mutation can lead to abnormal proliferation. To analyse the potential link betwen AP1 activity, encoded by members of the jun and fos gene families, and Ras-mediated cellular transformation, we have studied several NIH3T3 clones which overexpress the Ha-Ras or Ki-Ras oncogenes. These transformed ®broblasts accumulated higher levels of cJun, JunB, Fra1 and Fra2 proteins relative to their normal counterparts. They also displayed increased AP1 DNA binding activity which was predominantly composed of cJun and Fra1 containing dimers. Following serum stimulation of Ras clones, the elevated levels of cJun and Fra1 remained steady, while the induction of JunB and Fra2 was partially attenuated. Moreover, deregulated Ras signaling resulted in a complete loss of the serum inducibility of cFos and FosB. Ectopic co-expression of cJun and Fra1 in NIH3T3 ®broblasts led to a transformed phenotype, attenuation of cFos serum inducibility, increased AP1 activity and Cyclin D1 accumulation, all characteristics of oncogenic Ras expressing cells. These results demonstrate that cJun and Fra1 are crucial mediators of the Ras-transformation process.

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Section: Ras Enhances Ap1 Activitymentioning

confidence: 99%

Transformation by ras modifies AP1 composition and activity

et al. 1997

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“…Binding of the repressor to DNA was examined by the gel electrophoresis mobility shift assay [11][12][13]. Binding reactions were performed in 20/zl, as previously described [7].…”

Section: Dna-binding Assaymentioning

confidence: 99%

Interaction of BlaI, the repressor for the β‐lactamase gene of Bacillus licheniformis, with the blaP and blaI promoters

1989

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BlaI repressor for the fl-lactamase gene (blaP) of Bacillus licheniformis 749, was shown to repress expression of blaP and of the repressor gene (blal), using the purified protein in a DNA-directed in vitro translation assay. Binding of Blal to the promoter regions of blaP and blal was examined by equilibrium and competitive binding assays. BlaI binds to the blaP promoter with an equal or only slightly higher affinity than to the blal promoter. DNase I footprinting shows that BlaI binds directly to the blaP and blal promoters, such that RNA polymerase binding and/or transcript elongation would be blocked.

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“…Electrophoretic mobility shift assay (EMSA), developed by Fried and Crothers [1], and Garner and Revzin [2], is a popular method used for detection of protein-DNA interactions [3]. It is highly sensitive and may be used to obtain qualitative as well as quantitative information in determination of protein binding parameters of various DNA molecules [4][5][6].…”

Section: Introductionmentioning

confidence: 99%

Combination of Native and Denaturing PAGE for the Detection of Protein Binding Regions in Long Fragments of Genomic DNA

Kaer

Speek

2013

Methods in Molecular Biology

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Background: In a traditional electrophoresis mobility shift assay (EMSA) a 32 P-labeled doublestranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb) for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA (<10 kb) of known sequence.

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A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system

Cited by 1,966 publications

References 34 publications

Transformation by ras modifies AP1 composition and activity

Transformation by ras modifies AP1 composition and activity

Interaction of BlaI, the repressor for the β‐lactamase gene of Bacillus licheniformis, with the blaP and blaI promoters

Combination of Native and Denaturing PAGE for the Detection of Protein Binding Regions in Long Fragments of Genomic DNA

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