Nonradioactive gel mobility shift assay using chemiluminescent detection.
A nonradioactive gel mobility shift assay using chemiluminescent detection following semidry transfer to nylon membranes is described. The procedure utilizes digoxigenin-labeled oligonucleotides in conjunction with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase. Detection of alkaline phosphatase is by autoradiography of the chemiluminescence produced during enzymatic dephosphorylation of a dioxetane substrate. This method offers similar sensitivity to radioactive methods while having the advantages of multiple exposures, faster processing, stability of labeled oligonucleotides and safety associated with nonradioactive methods of detection.
Berger
R
R
Veterans Affairs Medical Center, Martinez, CA.
Duncan
M R
MR
Berman
B
B
D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D004076:Digoxigenin; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008163:Luminescent Measurements; D009687:Nuclear Proteins / Q000032:analysis* / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D004076:Digoxigenin