Electrophoretic mobility shift assay for the detection of specific DNA-protein complex in nuclear extracts from the cultured cells and frozen autopsy human brain tissue

Debomoy Lahiri, Yuan Wen Ge

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

The electrophoretic mobility shift assay (EMSA) is generally used to study the interaction of transcription factors to specific DNA sequences. The preparation of high quality nuclear extracts is an important step before performing the assay. Here we describe a rapid method for the isolation of good-quality DNA-binding proteins from cultured cell lines and autopsy tissue samples from the human brain. The 'rapid method' (RM) utilizes the low salt/detergent lysis steps followed by high salt extraction of nuclei. To test and compare the activity of nuclear extracts prepared by the standard and 'rapid' methods for its ability to form the specific DNA-protein complex, EMSA was carried out with three different oligonucleotide probes: AP1, NF-κB and URE. A comparative study indicates that the capacity to form the specific DNA-protein complex with these oligonucleotide probes by standard and RM nuclear extracts was very similar. Each nuclear extract formed the corresponding DNA-protein complex, the specificity of which was checked by the competition experiment. In some cases unspecific bands were observed and which were present in nuclear extracts from both preparations. Thus the simplicity of the 'rapid method' permits the preparation of nuclear extracts from several cell lines and tissue samples at the same time at much shorter time than the 'standard' method without compromising the DNA-binding activity. The RM can be applied to determine the cell type or tissue specificity of transcription factors in an efficient, economical and consistent manner. Copyright (C) 2000 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)257-265
Number of pages9
JournalBrain Research Protocols
Volume5
Issue number3
DOIs
StatePublished - Jul 2000

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Electrophoretic Mobility Shift Assay
Autopsy
Cultured Cells
DNA
Brain
Proteins
Oligonucleotide Probes
Transcription Factors
Salts
Cell Line
Organ Specificity
DNA-Binding Proteins
Detergents

Keywords

  • DNA-binding protein
  • Gel shift assay
  • Human brain tissue
  • Transcription factor

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

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N2 - The electrophoretic mobility shift assay (EMSA) is generally used to study the interaction of transcription factors to specific DNA sequences. The preparation of high quality nuclear extracts is an important step before performing the assay. Here we describe a rapid method for the isolation of good-quality DNA-binding proteins from cultured cell lines and autopsy tissue samples from the human brain. The 'rapid method' (RM) utilizes the low salt/detergent lysis steps followed by high salt extraction of nuclei. To test and compare the activity of nuclear extracts prepared by the standard and 'rapid' methods for its ability to form the specific DNA-protein complex, EMSA was carried out with three different oligonucleotide probes: AP1, NF-κB and URE. A comparative study indicates that the capacity to form the specific DNA-protein complex with these oligonucleotide probes by standard and RM nuclear extracts was very similar. Each nuclear extract formed the corresponding DNA-protein complex, the specificity of which was checked by the competition experiment. In some cases unspecific bands were observed and which were present in nuclear extracts from both preparations. Thus the simplicity of the 'rapid method' permits the preparation of nuclear extracts from several cell lines and tissue samples at the same time at much shorter time than the 'standard' method without compromising the DNA-binding activity. The RM can be applied to determine the cell type or tissue specificity of transcription factors in an efficient, economical and consistent manner. Copyright (C) 2000 Elsevier Science B.V.

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