A nonradioactive gel mobility shift assay using chemiluminescent detec
tion following semidry transfer to nylon membranes is described. The p
rocedure utilizes digoxigenin-labeled oligonucleotides in conjunction
with anti-digoxigenin antibody Fab fragments coupled to alkaline phosp
hatase. Detection of alkaline phosphatase is by autoradiography of the
chemiluminescence produced during enzymatic dephosphorylation of a di
oxetane substrate. This method offers similar sensitivity to radioacti
ve methods while having the advantages of multiple exposures, faster p
rocessing, stability of labeled oligonucleotides and safety associated
with nonradioactive methods of detection.