South Western blot mapping: A procedure for simultaneous characterization of DNA binding proteins and their specific genomic DNA target sites

Lelong, Jean‐Claude; Prevost, Grégoire; Lee, Kyong il; Crépin, Michel

doi:10.1016/0003-2697(89)90132-2

Cited by 19 publications

(9 citation statements)

References 15 publications

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“…After electrophoresis, the gel was equilibrated in transfer buffer (25mM Tris pH 8.3, 192mM glycine, 20~o (v/v) methanol, 0.1 ~o (w/v) SDS) for 30 min with gentle agitation and transferred using an AE-6670 Horizblot semi-dry electroblotting apparatus (ATTO Corporation, Tokyo, Japan). Southwestern blot analysis was performed based on methods developed by Bowen et al [5] and Lelong et al [27]. The blotted nitrocellulose membrane was washed twice for 15 min with 25 ml ofhybridisation buffer (25 mM HEPES-KOH pH 7.6, 4 0 m M KC1, 10~o (v/v)glycerol, i mM EDTA.Na2, 1 mM DTT, 0.02~o (w/v)bovine serum albumin, 0.02~o (w/v) Ficoll 400, 0.02 ~o (w/v) polyvinylpyrrolidone PVP-360) and then twice for 15 min with 25 ml of hybridisation buffer containing 30 #g/ml fish milt DNA.…”

Section: Southwestern Blot Analysismentioning

confidence: 99%

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter

1994

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Gel retardation assays using pea nuclear extracts have detected specific binding to regions of the promoter of the pea plastocyanin gene (petE). Several complexes which differ in sensitivity to competition with unlabelled promoter fragments and various DNA alternating copolymers, to heat treatment and to digestion with proteinase K have been detected. A protein factor, PCF1, forming one of these complexes was heat-stable and most sensitive to competition with poly(dAdT).poly(dAdT) compared to other alternating copolymers. DNase I footprinting assays showed that tracts of A/T-rich sequence within the -444 to -177 positive regulatory region of the petE promoter were protected in the presence of the pea nuclear extract. The factor PCF1 copurified with a high-mobility-group (HMG) protein preparation from pea chromatin. DNase I footprinting with the HMG protein preparation demonstrated that similar tracts of A/T-rich sequences within the promoter were protected. Southwestern-blot analysis of pea HMG proteins purified by gel filtration through Superose 12 detected a single DNA-binding species of 21 kDa. The properties of the factor PCF1 suggest that it is likely to be an HMG I protein.

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Section: Southwestern Blot Analysismentioning

confidence: 99%

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter

1994

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“…An assay combining Dot-Blot and ImmunoDetection (DBID) detects in vitro DNA-protein interactions experimenters' choice. We devised a simple assay to screen for inhibitors of protein-DNA interactions by borrowing the principles of Southwestern blotting [49,50] and immunochemistry [51,52] and applied it for CGGBP1.…”

Section: Resultsmentioning

confidence: 99%

An immunochemistry-based screen for chemical inhibitors of DNA-protein interactions and its application to human CGGBP1

Patel

Datta

et al. 2020

Preprint

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Background: Inhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins. Such a chemical inhibition of DNA-binding of proteins can be used to modulate processes such as replication, transcription, DNA repair and maintenance of epigenetic states. This prospect is currently challenged with the absence of robust and generic protocols to identify DNA-protein interactions. Additionally, much of the current approaches to designing inhibitors requires structural information of the target proteins.Methods: We have developed a simple dot blot and immunodetection-based assay to screen chemical libraries for inhibitors of DNA-protein interactions. The assay has been applied to a library of 1685 FDA-approved chemicals to discover inhibitors of CGGBP1, a multifunctional DNA-binding protein with no known structure. Additional in vitro and in cellulo assays have been performed to verify and supplement the findings of the screen.Results: Our primary screen has identified multiple inhibitors of direct or indirect interactions between CGGBP1 and genomic DNA. Of these, one inhibitor, Givinostat, was found to inhibit direct DNA-binding of CGGBP1 in the secondary screen using purified recombinant protein as the target. DNA and chromatin immunoprecipitation assays reinforced the findings of the screen that Givinostat inhibits CGGBP1-DNA binding.Conclusions: The assay we have described successfully identifies verifiable inhibitors of DNA-binding of protein; in this example, the human CGGBP1. This assay is customizable for a wide range of targets for which primary antibodies are available. It works with different sources of the target protein, cell lysates or purified recombinant preparations and does not require special equipment, DNA modifications or protein structural data. This assay is scalable and highly adaptable with the potential to discover inhibitors of transcription factors with implications in cancer biology.

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“…Nuclear extracts (10 µg) were run on a 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and electroblotted on nitrocellulose filters [13]. Blots were incubated for 2 h at room temperature in 1× binding buffer [see "Electrophoretic mobility shift assay (EMSA)"] in the presence of 5% dried milk powder and 1% DTT, subsequently for 2 h at room temperature in prehybridization solution (1× binding buffer supplemented with 0.25% dried milk powder, 1% DTT and 0.5 mg/ml herring sperm DNA).…”

Section: Southern-western Blot Analysismentioning

confidence: 99%

Characterization of the human type II Na/P i -cotransporter promoter

Hilfiker

Kvietikova

Hartmann

et al. 1998

Pfl�gers Archiv European Journal of Physiology

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The type II Na/Pi-cotransporter is expressed preferentially in renal proximal tubular epithelial cells. Comparison of the 5′ flanking region of the human NPT-2 gene with the opossum cell line (OK cell) and the murine Npt2 promoters revealed two conserved regions, one representing a putative C/EBP alpha site, the other a consensus TATA-box. In contrast to the OK cell and murine Npt-2 gene, the human exon 1 is flanked by two Alu-repeats, a short 90-bp inverted Alu element which is located within the promoter region and a full-length forward repeat present in intron 1. A 497-bp human promoter fragment including the inverted Alu-repeat was cloned in front of a luciferase reporter gene. The construct was active in OK and HeLa-S3 cells but no activity could be detected in the human monocyte cell line U937, the murine renal cortex cell line MCT and the dog kidney cell line MDCK. A twofold increase in promoter activity was observed in HeLa-S3 cells for a 5′ truncated fragment of 253 bp missing the inverted Alu-repeat. In the OK cell system the absence of the Alu-repeat was unable to modify promoter activity. In electrophoretic mobility shift assays (EMSAs) with a 31-bp oligonucleotide representing the conserved region with homology to C/EBP alpha we could provide evidence for specific DNA/protein interactions with nuclear extracts derived from kidney and liver cell lines but not for HeLa-S3 and U937 nuclear extracts. Specific interactions could also be observed with nuclear extracts from renal cortex, medulla and rat liver but not from rat spleen, intestine and heart. Southern-Western blotting techniques suggest that a 31-kDa nuclear protein from kidney-derived cells binds to the C/EBP-like region of the NPT2 promoter.

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South Western blot mapping: A procedure for simultaneous characterization of DNA binding proteins and their specific genomic DNA target sites

Cited by 19 publications

References 15 publications

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter

An immunochemistry-based screen for chemical inhibitors of DNA-protein interactions and its application to human CGGBP1

Characterization of the human type II Na/P i -cotransporter promoter

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