Transcription factor binding study by capillary zone electrophoretic mobility shift assay

Abstract

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.

Rapid qualitative evaluation of DNA transcription factor NF‐κB by microchip electrophoretic mobility shift assay in mammalian cells

“…For further characterization of the component of the dsOligo-NF-kB complex, a supershift assay might be possible by introducing an antibody, specific to the NF-kB, into the binding buffer. Capillary electrophoresis has already been used in integrating a supershift assay [25]. It will be necessary to establish a supershift assay as a future application of mEMSA to understand DNA-protein interactions.…”

“…There are several studies on electrophoretic mobility shift analysis by capillary electrophoresis [22][23][24][25]; however, reports using microfluidic chips are limited [26]. To evaluate the performance of mEMSA using a PMMA chip, we examined the migration times and peak areas of ssOligo, dsOligo (peaks 1, 2), and dsOligo-NF-kB complex (peak 3).…”

Bioanalytical interaction studies executed by preincubation affinity capillary electrophoresis

“…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].…”

Poly(ethylene oxide) facilitates the characterization of an affinity between strongly basic proteins with DNA by affinity capillary electrophoresis

“…The broad applicability of ACE methods to the study of binding interactions is illustrated by a variety of works on biomolecular interactions [11], such as protein-drug [12], protein-sugar [13], antibody-antigen [14], lectin-sugar [15], protein-or peptidepolymeric materials [16], drug-cyclodextrins [17,18], and drug-DNA [19]. Although many papers have reported ACE for protein-DNA interaction studies [20][21][22][23], very little literature describing dynamic complexation capillary electrophoresis (CE) has focused on these molecular interactions [24,25].…”