Electrophoretic mobility shift scanning using an automated infrared DNA sequencer.
Electrophoretic mobility shift assay (EMSA) is widely used in the study of sequence-specific DNA-binding proteins, including transcription factors and mismatch binding proteins. We have established a non-radioisotope-based protocol for EMSA that features an automated DNA sequencer with an infrared fluorescent dye (IRDye) detection unit. Our modification of the elec- trophoresis unit, which includes cooling the gel plates with a reduced well-to-read length, has made it possible to detect shifted bands within 1 h. Further, we have developed a rapid ligation-based method for generating IRDye-labeled probes with an approximately 60% cost reduction. This method has the advantages of real-time scanning, stability of labeled probes, and better safety associated with nonradioactive methods of detection. Analysis of a promoter from an industrially important filamentous fungus, Aspergillus oryzae, in a prototype experiment revealed that the method we describe has potential for use in systematic scanning and identification of the functionally important elements to which cellular factors bind in a sequence-specific manner.
Sano
M
M
National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan.
Ohyama
A
A
Takase
K
K
Yamamoto
M
M
Machida
M
M
D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay / Q000295:instrumentation*; D007259:Infrared Rays; D017422:Sequence Analysis, DNA / Q000295:instrumentation*
D004268:DNA-Binding Proteins; D004247:DNA