A fluorescence based non-radioactive electrophoretic mobility shift assay

Electrophoretic mobility shift assay (EMSA) or gel shift assay is one of th e most powerful methods for studying protein-DNA interactions. Typically, P -32-labeled DNA probes containing the sequence bound by the protein of inte rest are used in EMSA (rEMSA). Although rEMSA is sensitive and practicable, it relies on the handling of hazardous radioisotopes, and does not easily allow quantification. We developed a non-radioactive procedure using fluore scence (Cyano dye Cy5) labeled oligodeoxynucleotide duplexes as specific pr obes (fEMSA) and an automatic DNA sequencer for analysis. Testing different DNA-binding proteins (restriction endonuclease Eco RII, transcription fact or NF kappa B and it's subunit p50) the results in fEMSA and rEMSA are simi lar in regard to quality, reproducibility, and sensitivity. fEMSA allows a semiquantitative screening of large amounts of samples for specific DNA bin ding activities and is, therefore, a high throughput technology for semiqua ntitative analysis of DNA-protein interaction. (C) 2000 Elsevier Science B. V. All rights reserved.