Regulation and control of AP-1 binding activity in embryotoxicity.
The electrophoretic mobility shift assay (EMSA) is a sensitive relatively straightforward methodology used to detect sequence-specific DNA-protein interactions. It is the fundamental procedure of several variants that allow qualitative and quantitative assessments of protein-nucleic acid complexes. Classically, nuclear proteins and DNA are combined and the resulting mixture is electrophoretically separated in polyacrylamide or agarose gel under native conditions. The distribution within the gel is generally detected with autoradiography of the ³²P-labeled DNA. The underlying principle is that nucleic acid with protein bound to it will migrate more slowly through a gel matrix than the free nucleic acid. In this chapter, a representative protocol is described that addresses specific challenges of using whole embryos as the nuclear protein source, and the most common and informative EMSA variant, the "supershift," is also presented. The important points are underscored and approaches for troubleshooting are explained. References are provided for alternative methods and extensions of the basic protocol.
Department of Pharmacology and Toxicology, Queen's University, Kingston, ON, Canada. ozolinst@queensu.ca
Terence R S
TR
Ozolinš
D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts / Q000737:chemistry; D017364:Collagenases / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D004622:Embryo, Mammalian / Q000378:metabolism*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D051381:Rats; D020218:Response Elements; D013194:Staining and Labeling; D018808:Transcription Factor AP-1 / Q000737:chemistry / Q000378:metabolism*; D015017:Yolk Sac / Q000378:metabolism
D002457:Cell Extracts; D015342:DNA Probes; D010446:Peptide Fragments; D016760:Proto-Oncogene Proteins c-fos; D018808:Transcription Factor AP-1; D048031:JNK Mitogen-Activated Protein Kinases; D017364:Collagenases; C470319:Apex1 protein, rat; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase