Design and optimization of a capillary electrophoretic mobility shift assay involving trp repressor-DNA complexes

Stebbins, Michael; Hoyt, A.M.; Sepaniak, Michael J.; Hurlburt, Barry K.

doi:10.1016/0378-4347(96)00034-5

Cited by 41 publications

(39 citation statements)

References 35 publications

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“…Capillary versions of the gel-mobility shift assay, which also involves separation of protein-DNA complexes, have used buffers containing soluble polymers such as hydroxypropylmethylcellulose (HPMC) [28,29]. Such buffers may aid the separation by interacting with solutes and capillary walls to prevent adsorption.…”

Section: Effects Of Capillary Coating and Buffer Additivesmentioning

confidence: 99%

Effect of buffer, electric field, and separation time on detection of aptamer‐ligand complexes for affinity probe capillary electrophoresis

et al. 2003

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The separation and detection of complexes of aptamers and protein targets by capillary electrophoresis (CE) with laser-induced fluorescence was examined. Aptamer-thrombin and aptamer-immunoglobulin E (IgE) were used as model systems. Phosphate, 3-(N-morpholino)propanesulfonic acid with phosphate, and tris(hydroxyamino)methane-glycine-potassium (TGK) buffer at pH 8.4 were tested as electrophoresis media. Buffer had a large effect with TGK providing the most stable complexes for both protein-aptamer complexes. Conditions that suppressed electroosmotic flow, such as addition of hydroxypropylmethylcellulose to the media or modification of the capillary inner wall with polyacrylamide, were found to prevent detection of complexes. The effect of separation time and electric field were evaluated by monitoring complexes with electric field varied from 150-2850 V/cm and effective column lengths of 3.5 and 7.0 cm. As expected, shorter times on the column greatly increased peak heights for the complexes due to a combination of less dilution by diffusion and less dissociation on the column. High fields were found to have a detrimental effect on detection of complexes. It is concluded that the best conditions for detection of noncovalent complexes involve use of the minimal column length and electric field necessary to achieve separation. The results will be of interest in developing affinity probe CE assays wherein aptamers are used as affinity ligands.

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Section: Effects Of Capillary Coating and Buffer Additivesmentioning

confidence: 99%

Effect of buffer, electric field, and separation time on detection of aptamer‐ligand complexes for affinity probe capillary electrophoresis

et al. 2003

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“…Application of CE-based mobility shift assay for single-stranded DNA binding assay was also shown using laser-induced fluorescence polarization [5]. Previous investigations employed polymer additives to study of protein-DNA [6,7] and RNA-peptide [8] interactions. Recently, the use of uncoated fused-silica capillary with high electroosmotic flow was reported as suitable to DNA-protein interaction studies [9] and applied to the study of the MyoD and E47 transcription factors [10].…”

Section: Introductionmentioning

confidence: 99%

Transcription factor binding study by capillary zone electrophoretic mobility shift assay

Rónai

Khandurina

et al. 2003

Electrophoresis

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Regulation of gene expression through interaction of proteins with specific DNA sequences is a central issue in functional genomics. Capillary electrophoretic mobility shift assay is an efficient novel method for the investigation of sequence specific protein-DNA interactions, allowing rapid and sensitive quantification of the complex formation. In this paper, we present a pilot study on capillary zone electrophoretic mobility shift assay (CZEMSA) to investigate the interaction between the transcription factors of HeLa nuclear extract and Sp1-specific fluorescein-labeled oligonucleotide, using the unlabeled probe as competitor. The mobility shift assay was accomplished by CZE in coated capillaries without polymeric buffer additives. Specificity of the DNA protein complex formation was verified by competition experiments, as well as by supershift assay with an anti-Sp1 antibody. The applied electric field strength did not affect the stability of DNA-protein complex during the electrophoretic analysis, allowing rapid identification and quantification of the protein DNA interaction. A practical application to study the interaction between Oryza sativa MADS-box transcription factor 4 (OsMADS4) and its consensus sequence is also reported.

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“…The term CEMSA is often used in the field of DNA interactions, e.g. [19,20,[134][135][136][137]. We tend to prefer the term pre-eq CZE because the similarity between the names CEMSA and mobility shift ACE may lead to confusion.…”

Section: Interactions Involving Nucleotides Dna and Rnamentioning

confidence: 99%

“…A subsequent study investigated the relative affinities of polymorphic sequences of the 5' upstream region of DRD4 genes for their transcription factors using a competitive pre-eq CZE assay [135]. In an early feasibility study the interaction between trp operator (DNA) and trp repressor responsible for regulation of L-tryptophan biosynthesis was investigated [20]. Other examples related to the molecular biology of gene regulation are studies on DNA/oligonucleotide interactions with sea urchin embryo transcription factor [134], yeast transcriptional activator GCNK58 construct [138], MyoD and E47 transcription factors [139], histones H2B and H4 [136], ssDNA-binding protein [140], and serum response factor [141].…”

Section: Interactions Involving Nucleotides Dna and Rnamentioning

confidence: 99%

“…Different groups have used different names and acronyms for preeq CZE affinity studies making the literature less accessible. Among the terms that have been used for pre-eq CZE are CZE [14], equilibrium-mixture analysis [18], capillary electrophoretic mobility shift assay (CEMSA) [19,20], preincubation ACE (PI-CE) [21], and NECEEM for the variant introduced by Berezovski and Krylov [22,23]. We recommend using CE-FA as an acronym for CE conducted in the frontal analysis mode because the abbreviation FACE has been used for fluorescence anisotropy CE [7].…”

Section: Introductionmentioning

confidence: 99%

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Bioanalytical interaction studies executed by preincubation affinity capillary electrophoresis

Østergaard

Heegaard

2006

Electrophoresis

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The versatility of CE is beneficial for the study of many types of molecular interactions, because different experimental designs can be made to suit the characteristics of a particular interaction. A very versatile starting point is the preequilibration type of affinity CE that has been used extensively for characterizing biomolecular interactions in the last 15 years. We review this field here and include a comprehensive overview of the existing preincubation ACE modes including their advantages and limitations as well as the methodological developments and applications within the bioanalytical field.

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Design and optimization of a capillary electrophoretic mobility shift assay involving trp repressor-DNA complexes

Cited by 41 publications

References 35 publications

Effect of buffer, electric field, and separation time on detection of aptamer‐ligand complexes for affinity probe capillary electrophoresis

Effect of buffer, electric field, and separation time on detection of aptamer‐ligand complexes for affinity probe capillary electrophoresis

Transcription factor binding study by capillary zone electrophoretic mobility shift assay

Bioanalytical interaction studies executed by preincubation affinity capillary electrophoresis

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