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.
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-kappaB 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.
Lahiri
D K
DK
Laboratory of Molecular Neurogenetics, Institute of Psychiatric Research, Department of Psychiatry, Indiana University School of Medicine, 791 Union Drive, Indianapolis, IN 46202, USA. dlahiri@iupui.edu
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D020121:5' Untranslated Regions / Q000235:genetics; D033961:Adaptor Protein Complex 1; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D016564:Amyloid beta-Protein Precursor / Q000235:genetics; D000818:Animals; D001344:Autopsy; D001923:Brain Chemistry; D004247:DNA / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005615:Freezing; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics*; D016328:NF-kappa B / Q000032:analysis / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics; D015345:Oligonucleotide Probes; D016716:PC12 Cells; D010761:Phosphorus Radioisotopes; D051381:Rats
D020121:5' Untranslated Regions; D033961:Adaptor Protein Complex 1; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D016564:Amyloid beta-Protein Precursor; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D016328:NF-kappa B; D009419:Nerve Tissue Proteins; D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D004247:DNA