For EMSA labeled oligonucleotides are required, that include the specific target sequence. Radioactive labeling of the oligonucleotide is performed by kinase reaction (T4 kinase) using either 32P-ATP or 33P-ATP as a source for radioactivity. Due to the lower radiation load we recommend the use of 33P-ATP, which results in specific, detectable bands. Formation of a specific protein: DNA complex results in a lower mobility of this complex compared to the mobility of the free DNA. The specificity of the EMSA can also be analyzed by competition experiments in which an excess of unlabeled specific oligonucleotide interferes with the formation of a specific band, while the same amount of unrelated or mutated oligonucleotide has no effect. DNA-Protein complexes are then separated from the free (unbound) oligonucleotides by native gel-electrophoresis.
Swiss-Prot link Q05215: EGR-4 (human)